Novel cell-permeable succinate compounds

ABSTRACT

The present invention provides novel cell-permeable succinates and cell permeable precursors of succinate aimed at increasing ATP-production in mitochondria. The main part of ATP produced and utilized in the eukaryotic cell originates from mitochondrial oxidative phosphorylation, a process to which high-energy electrons are provided by the Kreb&#39;s cycle. Not all Kreb&#39;s cycle intermediates are readily permeable to the cellular membrane, one of them being succinate. The provision of the novel cell permeable succinates is envisaged to allow passage over the cellular membrane and thus the cell permeable succinates can be used to enhance mitochondrial ATP-output.

This application is a divisional of U.S. patent application Ser. No.15/128,480, filed Sep. 23, 2016, which is a § 371 application ofPCT/EP2015/057606, filed Apr. 8, 2015, which in turn claims priority toDK Application PA 2014 70190, filed Apr. 8, 2014. The entire disclosureof each of the foregoing applications is incorporated by referenceherein.

FIELD OF THE INVENTION

The present invention provides novel cell-permeable succinates and cellpermeable precursors of succinate aimed at increasing ATP-production inmitochondria. The main part of ATP produced and utilized in theeukaryotic cell originates from mitochondrial oxidative phosphorylation,a process to which high-energy electrons are provided by the Kreb'scycle. Not all Kreb's cycle intermediates are readily permeable to thecellular membrane, one of them being succinate. The provision of thenovel cell permeable succinates is envisaged to allow passage over thecellular membrane and thus the cell permeable succinates can be used toenhance mitochondrial ATP-output.

Moreover, present invention also provides for cell permeable succinatesor equivalents to succinates which in addition to being cell permeableand releasing succinate in the cytosol are also potentially able toprovide additional energy to the organism by the hydrolytic productsresulting from either chemical or enzymatic hydrolysis of the succinatederivatives.

The present invention also provides methods for preparing compounds ofthe invention that have improved properties for use in medicine and/orin cosmetics. Notably, the compounds of the invention are useful in theprevention or treatment of mitochondria-related disorders, inmaintaining normal mitochondrial function, enhancing mitochondrialfunction, i.e. producing more ATP than normally, or in restoring defectsin the mitochondrial respiratory system.

BACKGROUND OF THE INVENTION

Mitochondria are organelles in eukaryotic cells. They generate most ofthe cell's supply of adenosine triphosphate (ATP), which is used as anenergy source. Thus, mitochondria are indispensable for energyproduction, for the survival of eukaryotic cells and for correctcellular function. In addition to supplying energy, mitochondria areinvolved in a number of other processes such as cell signalling,cellular differentiation, cell death as well as the control of the cellcycle and cell growth. In particular, mitochondria are crucialregulators of cell apoptosis and they also play a major role in multipleforms of nonapoptotic cell death such as necrosis.

In recent years many papers have been published describing mitochondrialcontributions to a variety of diseases. Some diseases may be caused bymutations or deletions in the mitochondrial or nuclear genome, whileothers may be caused by primary or secondary impairment of themitochondrial respiratory system or other mechanisms related tomitochondrial dysfunction. At present there is no available treatmentthat can cure mitochondrial diseases.

In view of the recognized importance of maintaining or restoring anormal mitochondrial function or of enhancing the cell's energyproduction (ATP), there is a need to develop compounds which have thefollowing properties: Cell permeability of the parent, the ability toliberate intracellular succinate or a precursor of succinate, lowtoxicity of the parent compound and released products, andphysicochemical properties consistent with administration to a patient.

Succinate compounds have been prepared as prodrugs of other activeagents, for example WO 2002/28345 describes succinic acid bis(2,2-dimethylpropionyloxymethyl) ester, succinic acid dibutyryloxymethylester and succinic acid bis-(1-butyryloxyethyl)ester. These compoundsare prepared as agents to deliver formaldehyde, and are aimed atdifferent medical uses to the current compounds.

Prior art compounds include WO9747584, which describes a range of polyolsuccinates.

In the example given therein, Y is an H or alkyl group. Each succinatecompound contains multiple succinate moieties linked by a group ofstructure C(Y)—C(Q), and each ester acid is therefore directly linked toa moiety containing at least two carbon atoms in the form of an ethylgroup O—C—C. Each compound disclosed contains more than one succinatemoiety, and the succinate moiety is not protected by a moiety of typeO—C—X where X is a heteroatom.

Various succinate ester compounds are known in the art. Diethylsuccinate, monomethyl succinate and dimethyl succinate are shown to beinactive in the assays exemplified below, and fall outside the scope ofthe invention.

Moreover, U.S. Pat. No. 5,871,755 relates to dehydroalanine derivativesof succinamides for use as agents against oxidative stress and forcosmetic purposes.

DESCRIPTION OF THE INVENTION

A compound according to the invention is given by Formula (I)

or a pharmaceutically acceptable salt thereof, wherein the dotted bondbetween A and

B denotes an optional bond so as to form a ring closed structure, andwherein

Z is selected from —CH₂—CH₂— or >CH(CH₃),

A is selected from —SR, —OR and NHR, and R is

B is selected from —O—R′, —NHR″, —SR′″ or —OH; and R′ is selected fromthe formula (II) to (IX) below:

R′, R″ and R′″ are independently different or identical and is selectedfrom formula (IVVIII) below:

R₁ and R₃ are independently different or identical and are selected fromH, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, O-acyl, O-alkyl,N-acyl, N-alkyl, Xacyl, CH₂Xalkyl, CH₂X-acyl, F, CH₂COOH, CH₂CO₂alkyl,

X is selected from O, NH, NR₆, S,

R₂ is selected from Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl,C(O)CH₃, C(O)CH₂C(O)CH₃, C(O)CH₂CH(OH)CH₃,

p is an integer and is 1 or 2

R₆ is selected from H, Me, Et, propyl, i-propyl, butyl, iso-butyl,t-butyl, acetyl, acyl, propionyl, benzoyl, or formula (II), or formula(VIII)

X₅ is selected from —H, Me, Et, propyl, i-propyl, butyl, iso-butyl,t-butyl, —COOH, —C(═O)XR₆, CONR₁R₃ or is formula

X₇ is selected from R₁, —NR₁R₃,

R₉ is selected from H, Me, Et or O₂CCH₂CH₂COXR₈

R₁₀ is selected from Oacyl, NHalkyl, NHacyl, or O₂CCH₂CH₂COX₆R₈

X₆ is selected from O, NR₈, NR₆R₈, wherein R₆ and R₈ are independentlydifferent or identical and are is selected from H, alkyl, Me, Et,propyl, i-propyl, butyl, iso-butyl, t-butyl, acetyl, acyl, propionyl,benzoyl, or formula (II), or formula (VIII),

R₁₁ and R₁₂ are independently different or identical and are selectedfrom H, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, acetyl,propionyl, benzoyl, —CH₂Xalkyl, —CH₂Xacyl, where X is O, NR₆ or S,

R₁₁ and R₁₂ are independently different or identical and are selectedfrom CH₂Xalkyl, CH₂Xacyl, where X═O, NR₆ or S,

R₁₃, R₁₄ and R₁₅ are independently different or identical and areselected from H, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl,—COOH, O-acyl, O-alkyl, N-acyl, N-alkyl, Xacyl, CH₂Xalkyl;

Substituents on R13 and R14 or R13 and R15 may bridge to form a cyclicsystem to form cycloalkyl, heterocycloalkyl, lactone or lactams.

R_(f), R_(g) and R_(h) are independently different or identical and areselected from Xacyl, —CH₂Xalkyl, —CH₂X-acyl and R₉,

alkyl is selected from Me, Et, propyl, i-propyl, butyl, iso-butyl,t-butyl,

acyl is selected from formyl, acetyl, propionyl, isopropionyl, buturyl,tert-butyryl, pentanoyl, benzoyl, succinyl.

acyl and/or alkyl may be optionally substituted, and

when the dotted bond between A and B is present, the compound accordingto formula (I) is

wherein X₄ is selected from —COOH, —C(═O)XR₆,

The compounds of formula (I) (and any pharmaceutically acceptable saltsthereof) is referred to hereinafter as “compound of the invention”,“compounds of the invention” or as “compounds of the invention”.

Compounds of the invention of particular interest are those compoundswherein Z is —CH₂CH₂— and A is —SR.

Compounds of the invention of particular interest are those compounds,wherein Z is —CH₂CH₂—, A is SR, and B is OH or B is SR′″.

Compounds of the invention of particular interest are those compounds,wherein Z is —CH₂CH₂—, A is SR, B is OH or B is SR′″, where R′″ is

Compounds of the invention of particular interest are those compounds,wherein Z is —CH₂CH₂— and A is SR and B is OH.

Compounds of the invention of particular interest are those compounds,wherein Z is —CH₂CH₂—, A is SR, B is OH or B is SR, where R is

Compounds of the invention of particular interest are those compounds,wherein Z is —CH₂CH₂—, A is NR, B is OH and R is

and X is S.

Preferably, and with respect to formula (II), at least one of R₁ and R₃is —H, such that formula II is:

Preferably, and with respect to formula (VII), p=1 and X₅ is —H suchthat formula (VII) is

Preferably, and with respect to formula (VII), p=1 and X₅ is COXR₆ suchthat formula (VII) is

Preferably, and with respect to formula (VII), p=1 and X₅ is CONR₁R₃such that formula (VII) is

A compound according to formula (I) may be

wherein X₄ is selected from —COOH, —C(═O)XR₆,

Notably, a compound according to the invention is given by Formula (IA)

or a pharmaceutically acceptable salt thereof, wherein

Z is —CH₂—CH₂—,

A is selected from —SR, —OR and NHR, and R is

B is selected from —O—R′, —NHR″, —SR′″ or —OH; and

R′, R″ and R′″ are independently different or identical and is selectedfrom one or the formulas below:

R₁ and R₃ are independently different or identical and are selected fromH, Me, Et, propyl, O-Me, 0-Et, O-propyl,

X is selected from 0, NH, S,

p is an integer and is 1,

R₆ is selected from H, Me, Et,

X₆ is selected from —H, Me, Et, —COOH, —C(═O)XR₆, CONR₁R₃

X₇ is selected from R₁, —NR₁R₃,

R₁₃, R₁₄ and R₁₅ are independently different or identical and areselected from H, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl,—COOH, O-acyl, O-alkyl, N-acyl, N-alkyl, Xacyl, CH₂Xalkyl, wherein alkyland acyl are as defined herein before.

A compound of particular interest is given by Formula (IA)

or a pharmaceutically acceptable salt thereof, wherein

Z is —CH₂—CH₂—,

A is selected from —SR, —OR and NHR, and R is

B is selected from —O—R′, —NHR″, —SR′″ or —OH; and

R′, R″ and R′″ are independently different or identical and is selectedfrom one or the formulas below:

R₁ and R₃ are independently different or identical and are selected fromH, Me, Et, propyl, O-Me, O-Et, O-propyl,

X is selected from O, NH, S,

p is an integer and is 1,

R₆ is selected from H, Me, Et,

X₆ is selected from —H, Me, Et, —COOH, —C(═O)OR₆, CONR₁R₃,

X₇ is selected from R₁, —NR₁R₃,

R₁₃, R₁₄ and R₁₅ are independently different or identical and areselected from H, Me, Et, —COOH.

The following compound is known from Moore et al. J. Biol. Chem., 1982,257, pp. 10882-10892

However, the invention may or may not include these compounds for use intreatment of mitochondrial related diseases as discussed herein or forthe manufacture of a medicament for/in the treatment of mitochondrialrelated diseases as discussed herein.

Specific compounds according to the invention are:

General Chemistry Methods

The skilled person will recognise that the compounds of the inventionmay be prepared, in known manner, in a variety of ways. The routes beloware merely illustrative of some methods that can be employed for thesynthesis of compounds of formula (I). Compounds of the invention may bemade by starting with succinic acid, a mono-protected succinic acid, amono-activated methylmalonic acid a mono-protected methylmalonic acid ora mono-activated methylmalonic acid.

Protecting groups include but are not limited to benzyl and tert-butyl.Other protecting groups for carbonyls and their removal are detailed in‘Greene's Protective Groups in Organic Synthesis’ (Wuts and Greene,Wiley, 2006). Protecting groups may be removed by methods known to oneskilled in the art including hydrogenation in the presence of aheterogenous catalyst for benzyl esters and treatment with organic ormineral acids, preferably trifluoroacetic acid or dilute HCl, fortert-butyl esters.

Activating groups includes but is not limited to mixed anhydrides andacyl chlorides. Thus, were compounds of formula (I) are symmetrical thena symmetrical starting material is selected. Either a symmetricaldicarboxylic acid is selected or a di-activated carboxylic acid isselected. Preferably the compound selected is succinic acid or succinylchloride.

When the compound of formula (I) is asymmetric then the startingmaterial selected is asymmetric. That includes “acid-protected acid”,“acid-activated acid”, and “protected acid-activated acid”. Preferablythis includes succinic acid mono-benzyl ester, succinic acid mono-tertbutyl ester, 4-chloro-4-oxobutyric acid.

Alternatively for an asymmetric compound of formula (I) a symmetricstarting material is selected, preferable succinic acid, and lessderivatising starting material is employed.

The following general methods are not exhaustive and it will be apparentto one skilled in the art that other methods may be used to generatecompounds of the invention. The methods may be used together orseparately.

Compounds of formula (I) that contain formula (II) may be made byreacting a carboxylic acid with a suitable alkyl halide (formula (X)).E.g.

wherein Hal represents a halogen (e.g. F, Cl, Br or I) and R1, R2 and R3are as defined in formula (II). The reaction may conveniently be carriedout in a solvent such as dichloromethane, acetone, acetonitrile orN,N-dimethylformamide with a suitable base such as triethylamine,diisopropylethylamine or caesium carbonate at a temperature, forexample, in the range from −10° C. to 80° C., particularly at roomtemperature. The reaction may be performed with optional additives suchas sodium iodide or tetraalkyl ammonium halides (e.g. tetrabutylammonium iodide).

Compounds of formula X are either commercially available or may beconveniently prepared by literature methods such as those outlined inJournal of the American Chemical Society, 43, 660-7; 1921 or Journal ofmedicinal chemistry (1992), 35(4), 687-94.

Compounds of formula (I) that contain formula (VII) may be made byreacting an activated carboxylic acid with a compound of formula XIV,optionally in the presence of an activating species.

wherein X₅ and R₁ are as defined in formula (VII) and X₇ is Hal (Cl, F,Br) or mixed anhydride. Preferably X₇=Cl. The reaction may convenientlybe carried out in a solvent such as dichloromethane, acetone, THF,acetonitrile or N,N-dimethylformamide, with a suitable base such astriethylamine, diisopropylethylamine or caesium carbonate with at atemperature, for example, in the range from −10° C. to 80° C.,particularly at room temperature.

Compounds of formula (I) that contain formula (VIII) may be made byreacting an activated carboxylic acid with a compound of formula XIV,optionally in the presence of an activating species

wherein Hal represents a halogen (e.g. F, Cl, Br or I) and R₁₁, R₁₂ andR_(c) and R_(d) are as defined in formula (VIII). The reaction mayconveniently be carried out in a solvent such as dichloromethane,acetone, acetonitrile or N,N-dimethylformamide with a suitable base suchas triethylamine, diisopropylethylamine or caesium carbonate at atemperature, for example, in the range from −10° C. to 80° C.,particularly at 80° C. The reaction may be performed with optionaladditives such as sodium iodide or tetraalkyl ammonium halides (e.g.tetrabutyl ammonium iodide).

Compounds of formula X are either commercially available or may beconveniently prepared by literature methods whereby an amine is reactedwith an acyl chloride.

Compounds of formula (I) that contain formula (IX) may be made bycombining the methods describe above and by other methods known to oneskilled in the art.

General Use of the Compounds of the Invention

Compounds as described herein can be used in medicine or in cosmetics,or in the manufacture of a composition for such use. The medicament canbe used in any situation where an enhanced or restored energy production(ATP) is desired, such as in the treatment of metabolic diseases, or inthe treatment of diseases or conditions of mitochondrial dysfunction,treating or suppressing of mitochondrial disorders. The compounds may beused in the stimulation of mitochondrial energy production and in therestoration of drug-induced mitochondrial dysfunction such as e.g.sensineural hearing loss or tinnitus (side effect of certain antibioticsdue to mitochondrial toxicity) or lactic acidosis. The compounds may beused in the treatment of cancer, diabetes, acute starvation,endotoxemia, sepsis, systemic inflammatory response syndrome, multipleorgan dysfunction syndrome and following hypoxia, ischemia, stroke,myocardial infarction, acute angina, an acute kidney injury, coronaryocclusion and atrial fibrillation, or to avoid or counteract reperfusioninjuries. Moreover, it is envisaged that the compounds of the inventionmay be beneficial in treatment of male infertility.

It is envisaged that the compounds of the invention will providecell-permeable precursors of components of the Kreb's cycle andoptionally glycolysis pathways. It is envisaged that following entryinto the cell, enzymatic or chemical hydrolysis will liberate succinateor methylmalonate optionally along with other energy-providingmaterials, such as acetate and glucose.

The compounds of the invention can be used to enhance or restore energyproduction in mitochondria. Notably the compounds can be used inmedicine or in cosmetics. The compounds can be used in the prevention ortreatment of disorders or diseases having a component relating tomitochondrial dysfunction and/or to a component of energy (ATP)deficiency.

Enhancement of energy production is e.g. relevant in subjects sufferingfrom a mitochondrial defect, disorder or disease. Mitochondrial diseasesresult from dysfunction of the mitochondria, which are specializedcompartments present in every cell of the body except red blood cells.When mitochondrial function decreases, the energy generated within thecell reduces and cell injury or cell death will follow. If this processis repeated throughout the body the life of the subject is severelycompromised.

Diseases of the mitochondria appear most often in organs that are veryenergy demanding such as retina, the cochlea, the brain, heart, liver,skeletal muscles, kidney and the endocrine and respiratory system.

Symptoms of a mitochondrial disease may include loss of motor control,muscle weakness and pain, seizures, visual/hearing problems, cardiacdiseases, liver diseases, gastrointestinal disorders, swallowingdifficulties and more.

A mitochondrial disease may be inherited or may be due to spontaneousmutations, which lead to altered functions of the proteins or RNAmolecules normally residing in the mitochondria.

Many diseases have been found to involve a mitochondrial deficiency suchas a Complex I, II, III or IV deficiency or an enzyme deficiency likee.g. pyruvate dehydrogenase deficiency. However, the picture is complexand many factors may be involved in the diseases.

Up to now, no curative treatments are available. The only treatmentsavailable are such that can alleviate the symptoms and delay theprogression of the disease. Accordingly, the findings by the presentinventors and described herein are very important as they demonstratethe beneficial effect of the cell permeable compounds of succinic acidon the energy production in the mitochondria.

In addition, in comparison with known succinate prodrugs (such as e.g.mentioned in WO 97/47584), they show improved properties for treatmentof these and related diseases, including better cell permeability,longer plasma half-life, reduced toxicity, increased energy release tomitochondria, and improved formulation (due to improved propertiesincluding increased solubility). In some cases, the compounds are alsoorally bioavailable, which allows for easier administration.

Thus the advantageous properties of the compound of the invention mayinclude one or more of the following:

-   -   Increased cell permeability    -   Longer half-life in plasma    -   Reduced toxicity    -   Increased energy release to mitochondria    -   Improved formulation    -   Increased solubility    -   Increased oral bioavailability

The present invention provides the compound of the invention for use asa pharmaceutical, in particular in the treatment of cellular energy(ATP)-deficiency.

A compound of the invention may be used in the treatment of complex Iimpairment, either dysfunction of the complex itself or any condition ordisease that limits the supply of NADH to Complex I, e.g. dysfunction ofKrebs cycle, glycolysis, beta-oxidation, pyruvate metabolism and eventransport of glucose or other Complex-I-related substrates).

The present invention also provides a method of treatment ofmitochondrial complex I related disorders such as but not limited to,Leigh Syndrome, Leber's hereditary optic neuropathy (LHON), MELAS(mitochondrial encephalomyopathy, lactic acidosis, and stroke-likeepisodes) and MERRF (myoclonic epilepsy with ragged red fibers), whichcomprises administering to a subject in need thereof an effective amountof the compound of the invention.

The present invention also provides the use of the compound of theinvention for the manufacture of a medicament for the treatment ofdrug-induced lactic acidosis.

A compound of the invention may also be useful in any condition whereextra energy production would potentially be beneficial such as, but notlimited to, prolonged surgery and intensive care.

Mitochondria

Mitochondria are organelles in eukaryotic cells, popularly referred toas the “powerhouse” of the cell. One of their primary functions isoxidative phosphorylation. The molecule adenosine triphosphate (ATP)functions as an energy “currency” or energy carrier in the cell, andeukaryotic cells derive the majority of their ATP from biochemicalprocesses carried out by mitochondria. These biochemical processesinclude the citric acid cycle (the tricarboxylic acid cycle, or Kreb'scycle), which generates reduced nicotinamide adenine dinucleotide (NADH)from oxidized nicotinamide adenine dinucleotide (NAD⁺) and reducedflavin adenine dinucleotide (FADH2) from oxidized flavin adeninedinucleotide (FAD), as well as oxidative phosphorylation, during whichNADH and FADH2 is oxidized back to NAD^(<+>) and FAD.

The electrons released by oxidation of NADH are shuttled down a seriesof protein complexes (Complex I, Complex II, Complex III, and ComplexIV) known as the respiratory chain. The oxidation of succinate occurs atComplex II (succinate dehydrogenase complex) and FAD is a prostheticgroup in the enzyme complex succinate dehydrogenase (complex II). Therespiratory complexes are embedded in the inner membrane of themitochondrion. Complex IV, at the end of the chain, transfers theelectrons to oxygen, which is reduced to water. The energy released asthese electrons traverse the complexes is used to generate a protongradient across the inner membrane of the mitochondrion, which createsan electrochemical potential across the inner membrane. Another proteincomplex, Complex V (which is not directly associated with Complexes I,II, III and IV) uses the energy stored by the electrochemical gradientto convert ADP into ATP.

The citric acid cycle and oxidative phosphorylation are preceded byglycolysis, in which a molecule of glucose is broken down into twomolecules of pyruvate, with net generation of two molecules of ATP permolecule of glucose. The pyruvate molecules then enter the mitochondria,where they are completely oxidized to CO₂ and H₂O via oxidativephosphorylation (the overall process is known as aerobic respiration).The complete oxidation of the two pyruvate molecules to carbon dioxideand water yields about at least 28-29 molecules of ATP, in addition tothe 2 molecules of ATP generated by transforming glucose into twopyruvate molecules. If oxygen is not available, the pyruvate moleculedoes not enter the mitochondria, but rather is converted to lactate, inthe process of anaerobic respiration.

The overall net yield per molecule of glucose is thus approximately atleast 30-31 ATP molecules. ATP is used to power, directly or indirectly,almost every other biochemical reaction in the cell. Thus, the extra(approximately) at least 28 or 29 molecules of ATP contributed byoxidative phosphorylation during aerobic respiration are critical to theproper functioning of the cell. Lack of oxygen prevents aerobicrespiration and will result in eventual death of almost all aerobicorganisms; a few organisms, such as yeast, are able to survive usingeither aerobic or anaerobic respiration.

When cells in an organism are temporarily deprived of oxygen, anaerobicrespiration is utilized until oxygen again becomes available or the celldies. The pyruvate generated during glycolysis is converted to lactateduring anaerobic respiration. The build-up of lactic acid is believed tobe responsible for muscle fatigue during intense periods of activity,when oxygen cannot be supplied to the muscle cells. When oxygen againbecomes available, the lactate is converted back into pyruvate for usein oxidative phosphorylation.

Mitochondrial dysfunction contributes to various disease states. Somemitochondrial diseases are due to mutations or deletions in themitochondrial genome or nuclear. If a threshold proportion ofmitochondria in the cell are defective, and if a threshold proportion ofsuch cells within a tissue have defective mitochondria, symptoms oftissue or organ dysfunction can result. Practically any tissue can beaffected, and a large variety of symptoms may be present, depending onthe extent to which different tissues are involved.

Use of the Compounds of the Invention

The compounds of the invention may be used in any situation where anenhanced or restored energy production (ATP) is desired. Examples aree.g. in all clinical conditions where there is a potential benefit ofincreased mitochondrial ATP-production or a restoration of mitochondrialfunction, such as in the restoration of drug-induced mitochondrialdysfunction or lactic acidosis and the treatment of cancer, diabetes,acute starvation, endotoxemia, sepsis, reduced hearing visual acuity,systemic inflammatory response syndrome and multiple organ dysfunctionsyndrome. The compounds may also be useful following hypoxia, ischemia,stroke, myocardial infarction, acute angina, an acute kidney injury,coronary occlusion, atrial fibrillation and in the prevention orlimitations of reperfusion injuries.

In particular, the compounds of the invention can be used in medicine,notably in the treatment or prevention of a mitochondria-relatedcondition, disease or disorder or in cosmetics.

Dysfunction of mitochondria is also described in relation to renaltubular acidosis; motor neuron diseases; other neurological diseases;epilepsy; genetic diseases; Huntington's Disease; mood disorders;schizophrenia; bipolar disorder; age-associated diseases; cerebralvascular accidents, macular degeneration; diabetes; and cancer.

Compounds of the invention for use in mitochondrial related disorders ordiseases The compounds according to the invention may be used in theprevention or treatment a mitochondria-related disease selected from thefollowing:

-   -   Alpers Disease (Progressive Infantile Poliodystrophy)    -   Amyotrophic lateral sclerosis (ALS)    -   Autism    -   Barth syndrome (Lethal Infantile Cardiomyopathy)    -   Beta-oxidation Defects    -   Bioenergetic metabolism deficency    -   Carnitine-Acyl-Carnitine Deficiency    -   Carnitine Deficiency    -   Creatine Deficiency Syndromes (Cerebral Creatine Deficiency        Syndromes (CCDS) includes: Guanidinoaceteate Methyltransferase        Deficiency (GAMT Deficiency), L-Arginine: Glycine        Amidinotransferase Deficiency (AGAT Deficiency), and        SLC6A8-Related Creatine Transporter Deficiency (SLC6A8        Deficiency).    -   Co-Enzyme Q10 Deficiency    -   Complex I Deficiency (NADH dehydrogenase (NADH-CoQ reductase)        deficiency)    -   Complex II Deficiency (Succinate dehydrogenase deficiency)    -   Complex III Deficiency (Ubiquinone-cytochrome c oxidoreductase        deficiency)    -   Complex IV Deficiency/COX Deficiency (Cytochrome c oxidase        deficiency is caused by a defect in Complex IV of the        respiratory chain)    -   Complex V Deficiency (ATP synthase deficiency)    -   COX Deficiency    -   CPEO (Chronic Progressive External Ophthalmoplegia Syndrome)    -   CPT I Deficiency    -   CPT II Deficiency    -   Friedreich's ataxia (FRDA or FA)    -   Glutaric Aciduria Type II    -   KSS (Kearns-Sayre Syndrome)    -   Lactic Acidosis    -   LCAD (Long-Chain Acyl-CoA Dehydrogenase Deficiency)    -   LCHAD    -   Leigh Disease or Syndrome (Subacute Necrotizing        Encephalomyelopathy)    -   LHON (Leber's hereditary optic neuropathy)    -   Luft Disease    -   MCAD (Medium-Chain Acyl-CoA Dehydrogenase Deficiency)    -   MELAS (Mitochondrial Encephalomyopathy Lactic Acidosis and        Strokelike Episodes)    -   MERRF (Myoclonic Epilepsy and Ragged-Red Fiber Disease)    -   MIRAS (Mitochondrial Recessive Ataxia Syndrome)    -   Mitochondrial Cytopathy    -   Mitochondrial DNA Depletion    -   Mitochondrial Encephalopathy includes: Encephalomyopathy,        Encephalomyelopathy    -   Mitochondrial Myopathy    -   MNGIE (Myoneurogastointestinal Disorder and Encephalopathy)    -   NARP (Neuropathy, Ataxia, and Retinitis Pigmentosa)    -   Neurodegenerative disorders associated with Parkinson's,        Alzheimer's or Huntington's disease    -   Pearson Syndrome    -   Pyruvate Carboxylase Deficiency    -   Pyruvate Dehydrogenase Deficiency    -   POLG Mutations    -   Respiratory Chain Deficiencies    -   SCAD (Short-Chain Acyl-CoA Dehydrogenase Deficiency)    -   SCHAD (Short Chain L-3-Hydroxyacyl-CoA Dehydrogenase (SCHAD)        Deficiency, also referred to as 3-Hydroxy Acyl CoA Dehydrogenase        Deficiency HADH    -   VLCAD (Very Long-Chain Acyl-CoA Dehydrogenase Deficiency)    -   Diabetes    -   Acute starvation    -   Endotoxemia    -   Sepsis    -   Systemic inflammation response syndrome (SIRS)    -   Multiple organ failure

With reference to information from the web-page of United MitochondrialDisease Foundation (www.umdf.org), some of the above-mentioned diseasesare discussed in more details in the following:

Complex I deficiency: Inside the mitochondrion is a group of proteinsthat carry electrons along four chain reactions (Complexes I-IV),resulting in energy production. This chain is known as the ElectronTransport Chain. A fifth group (Complex V) churns out the ATP. Together,the electron transport chain and the ATP synthase form the respiratorychain and the whole process is known as oxidative phosphorylation orOXPHOS.

Complex I, the first step in this chain, is the most common site formitochondrial abnormalities, representing as much as one third of therespiratory chain deficiencies. Often presenting at birth or in earlychildhood, Complex I deficiency is usually a progressiveneurodegenerative disorder and is responsible for a variety of clinicalsymptoms, particularly in organs and tissues that require high energylevels, such as brain, heart, liver, and skeletal muscles. A number ofspecific mitochondrial disorders have been associated with Complex Ideficiency including: Leber's hereditary optic neuropathy (LHON), MELAS,MERRF, and Leigh Syndrome (LS). MELAS stands for (mitochondrialencephalomyopathy, lactic acidosis, and stroke-like episodes) and MERRFstand for myoclonic epilepsy with ragged red fibers.

LHON is characterized by blindness which occurs on average between 27and 34 years of age; blindness can develop in both eyes simultaneously,or sequentially (one eye will develop blindness, followed by the othereye two months later on average).

Other symptoms may also occur, such as cardiac abnormalities andneurological complications.

There are three major forms of Complex I deficiency:

i) Fatal infantile multisystem disorder—characterized by poor muscletone, developmental delay, heart disease, lactic acidosis, andrespiratory failure.

ii) Myopathy (muscle disease)—starting in childhood or adulthood, andcharacterized by weakness or exercise intolerance.

iii) Mitochondrial encephalomyopathy (brain and muscledisease)—beginning in childhood or adulthood and involving variablesymptom combinations which may include: eye muscle paralysis, pigmentaryretinopathy (retinal color changes with loss of vision), hearing loss,sensory neuropathy (nerve damage involving the sense organs), seizures,dementia, ataxia (abnormal muscle coordination), and involuntarymovements. This form of Complex I deficiency may cause Leigh Syndromeand MELAS.

Most cases of Complex I deficiency result from autosomal recessiveinheritance (combination of defective nuclear genes from both the motherand the father). Less frequently, the disorder is maternally inheritedor sporadic and the genetic defect is in the mitochondrial DNA.

Treatment: As with all mitochondrial diseases, there is presently nocure for Complex I deficiency. A variety of treatments, which may or maynot be effective, can include such metabolic therapies as: riboflavin,thiamine, biotin, co-enzyme Q10, carnitine, and ketogenic diet.Therapies for the infantile multisystem form have been unsuccessful.

The clinical course and prognosis for Complex I patients is highlyvariable and may depend on the specific genetic defect, age of onset,organs involved, and other factors.

Complex III Deficiency: The symptoms include four major forms:

i) Fatal infantile encephalomyopathy, congenital lactic acidosis,hypotonia, dystrophic posturing, seizures, and coma. Ragged-red fibersin muscle tissue are common.

ii) Encephalomyopathies of later onset (childhood to adult life):various combinations of weakness, short stature, ataxia, dementia,hearing loss, sensory neuropathy, pigmentary retinopathy, and pyramidalsigns. Ragged-red fibers are common. Possible lactic acidosis.

iii) Myopathy, with exercise intolerance evolving into fixed weakness.Ragged-red fibers are common. Possible lactic acidosis.

iv) Infantile histiocytoid cardiomyopathy.

Complex IV Deficiency/COX Deficiency. The symptoms include two majorforms:

-   -   1. Encephalomyopathy: Typically normal for the first 6 to 12        months of life and then show developmental regression, ataxia,        lactic acidosis, optic atrophy, ophthalmoplegia, nystagmus,        dystonia, pyramidal signs, and respiratory problems. Frequent        seizures. May cause Leigh Syndrome        -   2. Myopathy: Two main variants:        -   1. Fatal infantile myopathy: may begin soon after birth and            accompanied by hypotonia, weakness, lactic acidosis,            ragged-red fibers, respiratory failure, and kidney problems.        -   2. Benign infantile myopathy: may begin soon after birth and            accompanied by hypotonia, weakness, lactic acidosis,            ragged-red fibers, respiratory problems, but (if the child            survives) followed by spontaneous improvement.

KSS (Kearns-Sayre Syndrome): KSS is a slowly progressive multi-systemmitochondrial disease that often begins with drooping of the eyelids(ptosis). Other eye muscles eventually become involved, resulting inparalysis of eye movement. Degeneration of the retina usually causesdifficulty seeing in dimly lit environments.

KSS is characterized by three main features:

-   -   typical onset before age 20 although may occur in infancy or        adulthood    -   paralysis of specific eye muscles (called chronic progressive        external ophthalmoplegia—CPEO)    -   degeneration of the retina causing abnormal accumulation of        pigmented (colored) material (pigmentary retinopathy).

In addition, one or more of the following conditions is present:

-   -   block of electrical signals in the heart (cardiac conduction        defects)    -   elevated cerebrospinal fluid protein    -   incoordination of movements (ataxia).

Patients with KSS may also have such problems as deafness, dementia,kidney dysfunction, and muscle weakness. Endocrine abnormalitiesincluding growth retardation, short stature, or diabetes may also beevident.

KSS is a rare disorder. It is usually caused by a single large deletion(loss) of genetic material within the DNA of the mitochondria (mtDNA),rather than in the DNA of the cell nucleus. These deletions, of whichthere are over 150 species, typically arise spontaneously. Lessfrequently, the mutation is transmitted by the mother.

As with all mitochondrial diseases, there is no cure for KSS.

Treatments are based on the types of symptoms and organs involved, andmay include: Coenzyme Q10, insulin for diabetes, cardiac drugs, and acardiac pacemaker which may be life-saving. Surgical intervention fordrooping eyelids may be considered but should be undertaken byspecialists in ophthalmic surgical centers.

KSS is slowly progressive and the prognosis varies depending onseverity. Death is common in the third or fourth decade and may be dueto organ system failures.

Leigh Disease or Syndrome (Subacute Necrotizing Encephalomyelopathy):Symptoms: Seizures, hypotonia, fatigue, nystagmus, poor reflexes, eatingand swallowing difficulties, breathing problems, poor motor function,ataxia.

Causes: Pyruvate Dehydrogenase Deficiency, Complex I Deficiency, ComplexII Deficiency, Complex IV/COX Deficiency, NARP.

Leigh's Disease is a progressive neurometabolic disorder with a generalonset in infancy or childhood, often after a viral infection, but canalso occur in teens and adults. It is characterized on MRI by visiblenecrotizing (dead or dying tissue) lesions on the brain, particularly inthe midbrain and brainstem.

The child often appears normal at birth but typically begins displayingsymptoms within a few months to two years of age, although the timingmay be much earlier or later. Initial symptoms can include the loss ofbasic skills such as sucking, head control, walking and talking. Thesemay be accompanied by other problems such as irritability, loss ofappetite, vomiting and seizures. There may be periods of sharp declineor temporary restoration of some functions. Eventually, the child mayalso have heart, kidney, vision, and breathing complications.

There is more than one defect that causes Leigh's Disease. These includea pyruvate dehydrogenase (PDHC) deficiency, and respiratory chain enzymedefects—Complexes I, II, IV, and V. Depending on the defect, the mode ofinheritance may be X-linked dominant (defect on the X chromosome anddisease usually occurs in males only), autosomal recessive (inheritedfrom genes from both mother and father), and maternal (from motheronly). There may also be spontaneous cases which are not inherited atall.

There is no cure for Leigh's Disease. Treatments generally involvevariations of vitamin and supplement therapies, often in a “cocktail”combination, and are only partially effective. Various resource sitesinclude the possible usage of: thiamine, coenzyme Q10, riboflavin,biotin, creatine, succinate, and idebenone. Experimental drugs, such asdichloroacetate (DCA) are also being tried in some clinics. In somecases, a special diet may be ordered and must be monitored by adietitian knowledgeable in metabolic disorders.

The prognosis for Leigh's Disease is poor. Depending on the defect,individuals typically live anywhere from a few years to the mid-teens.Those diagnosed with Leigh-like syndrome or who did not display symptomsuntil adulthood tend to live longer.

MELAS (Mitochondrial Encephalomyopathy Lactic Acidosis and Stroke-likeEpisodes): Symptoms: Short statue, seizures, stroke-like episodes withfocused neurological deficits, recurrent headaches, cognitiveregression, disease progression, ragged-red fibers.

Cause: Mitochondrial DNA point mutations: A3243G (most common)MELAS—Mitochondrial Myopathy (muscle weakness), Encephalopathy (brainand central nervous system disease), Lactic Acidosis (build-up of aproduct from anaerobic respiration), and Stroke-like episodes (partialparalysis, partial vision loss, or other neurological abnormalities).

MELAS is a progressive neurodegenerative disorder with typical onsetbetween the ages of 2 and 15, although it may occur in infancy or aslate as adulthood. Initial symptoms may include stroke-like episodes,seizures, migraine headaches, and recurrent vomiting.

Usually, the patient appears normal during infancy, although shortstature is common. Less common are early infancy symptoms that mayinclude developmental delay, learning disabilities or attention-deficitdisorder. Exercise intolerance, limb weakness, hearing loss, anddiabetes may also precede the occurrence of the stroke-like episodes.

Stroke-like episodes, often accompanied by seizures, are the hallmarksymptom of MELAS and cause partial paralysis, loss of vision, and focalneurological defects. The gradual cumulative effects of these episodesoften result in variable combinations of loss of motor skills (speech,movement, and eating), impaired sensation (vision loss and loss of bodysensations), and mental impairment (dementia). MELAS patients may alsosuffer additional symptoms including: muscle weakness, peripheral nervedysfunction, diabetes, hearing loss, cardiac and kidney problems, anddigestive abnormalities. Lactic acid usually accumulates at high levelsin the blood, cerebrospinal fluid, or both. MELAS is maternallyinherited due to a defect in the DNA within mitochondria. There are atleast 17 different mutations that can cause MELAS. By far the mostprevalent is the A3243G mutation, which is responsible for about 80% ofthe cases.

There is no cure or specific treatment for MELAS. Although clinicaltrials have not proven their efficacy, general treatments may includesuch metabolic therapies as: CoQ10, creatine, phylloquinone, and othervitamins and supplements. Drugs such as seizure medications and insulinmay be required for additional symptom management. Some patients withmuscle dysfunction may benefit from moderate supervised exercise. Inselect cases, other therapies that may be prescribed includedichloroacetate (DCA) and menadione, though these are not routinely useddue to their potential for having harmful side effects.

The prognosis for MELAS is poor. Typically, the age of death is between10 to 35 years, although some patients may live longer. Death may comeas a result of general body wasting due to progressive dementia andmuscle weakness, or complications from other affected organs such asheart or kidneys.

MERRF is a progressive multi-system syndrome usually beginning inchildhood, but onset may occur in adulthood. The rate of progressionvaries widely. Onset and extent of symptoms can differ among affectedsiblings.

The classic features of MERRF include:

-   -   Myoclonus (brief, sudden, twitching muscle spasms)—the most        characteristic symptom    -   Epileptic seizures    -   Ataxia (impaired coordination)    -   Ragged-red fibers (a characteristic microscopic abnormality        observed in muscle biopsy of patients with MERRF and other        mitochondrial disorders) Additional symptoms may include:        hearing loss, lactic acidosis (elevated lactic acid level in the        blood), short stature, exercise intolerance, dementia, cardiac        defects, eye abnormalities, and speech impairment.

Although a few cases of MERRF are sporadic, most cases are maternallyinherited due to a mutation within the mitochondria. The most commonMERRF mutation is A8344G, which accounted for over 80% of the cases.Four other mitochondrial DNA mutations have been reported to causeMERRF. While a mother will transmit her MERRF mutation to all of heroffspring, some may never display symptoms.

As with all mitochondrial disorders, there is no cure for MERRF.Therapies may include coenzyme Q10, L-carnitine, and various vitamins,often in a “cocktail” combination. Management of seizures usuallyrequires anticonvulsant drugs. Medications for control of other symptomsmay also be necessary.

The prognosis for MERRF varies widely depending on age of onset, typeand severity of symptoms, organs involved, and other factors.

Mitochondrial DNA Depletion: The symptoms include three major forms:

1. Congenital myopathy: Neonatal weakness, hypotonia requiring assistedventilation, possible renal dysfunction. Severe lactic acidosis.Prominent ragged-red fibers. Death due to respiratory failure usuallyoccurs prior to one year of age.

2. Infantile myopathy: Following normal early development until one yearold, weakness appears and worsens rapidly, causing respiratory failureand death typically within a few years.

3. Hepatopathy: Enlarged liver and intractable liver failure, myopathy.Severe lactic acidosis. Death is typical within the first year.

Friedreich's Ataxia

Friedreich's ataxia (FRDA or FA) an autosomal recessiveneurodegenerative and cardiodegenerative disorder caused by decreasedlevels of the protein frataxin. Frataxin is important for the assemblyof iron-sulfur clusters in mitochondrial respiratory-chain complexes.Estimates of the prevalence of FRDA in the United States range from 1 inevery 22,000-29,000 people (seewww.nlm.nih.gov/medlineplus/ency/article/001411.htm) to 1 in 50,000people. The disease causes the progressive loss of voluntary motorcoordination (ataxia) and cardiac complications. Symptoms typicallybegin in childhood, and the disease progressively worsens as the patientgrows older; patients eventually become wheelchair-bound due to motordisabilities.

In addition to congenital disorders involving inherited defectivemitochondria, acquired mitochondrial dysfunction has been suggested tocontribute to diseases, particularly neurodegenerative disordersassociated with aging like Parkinson's, Alzheimer's, and Huntington'sDiseases. The incidence of somatic mutations in mitochondrial DNA risesexponentially with age; diminished respiratory chain activity is founduniversally in aging people. Mitochondrial dysfunction is alsoimplicated in excitotoxicity, neuronal injury, cerebral vascularaccidents such as that associated with seizures, stroke and ischemia.

Pharmaceutical Compositions Comprising a Compound of the Invention

The present invention also provides a pharmaceutical compositioncomprising the compound of the invention together with one or morepharmaceutically acceptable diluents or carriers.

The compound of the invention or a formulation thereof may beadministered by any conventional method for example but withoutlimitation it may be administered parenterally, orally, topically(including buccal, sublingual or transdermal), via a medical device(e.g. a stent), by inhalation or via injection (subcutaneous orintramuscular). The treatment may consist of a single dose or aplurality of doses over a period of time.

The treatment may be by administration once daily, twice daily, threetimes daily, four times daily etc. The treatment may also be bycontinuous administration such as e.g. administration intravenous bydrop.

Whilst it is possible for the compound of the invention to beadministered alone, it is preferable to present it as a pharmaceuticalformulation, together with one or more acceptable carriers. Thecarrier(s) must be “acceptable” in the sense of being compatible withthe compound of the invention and not deleterious to the recipientsthereof. Examples of suitable carriers are described in more detailbelow.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.Such methods include the step of bringing into association the activeingredient (compound of the invention) with the carrier whichconstitutes one or more accessory ingredients. In general theformulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers or finely dividedsolid carriers or both, and then, if necessary, shaping the product.

The compound of the invention will normally be administeredintravenously, orally or by any parenteral route, in the form of apharmaceutical formulation comprising the active ingredient, optionallyin the form of a non-toxic organic, or inorganic, acid, or base,addition salt, in a pharmaceutically acceptable dosage form. Dependingupon the disorder and patient to be treated, as well as the route ofadministration, the compositions may be administered at varying doses.

The pharmaceutical compositions must be stable under the conditions ofmanufacture and storage; thus, preferably should be preserved againstthe contaminating action of microorganisms such as bacteria and fungi.The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (e.g. glycerol, propylene glycol andliquid polyethylene glycol), vegetable oils, and suitable mixturesthereof.

For example, the compound of the invention can also be administeredorally, buccally or sublingually in the form of tablets, capsules,ovules, elixirs, solutions or suspensions, which may contain flavouringor colouring agents, for immediate-, delayed- or controlled-releaseapplications.

Formulations in accordance with the present invention suitable for oraladministration may be presented as discrete units such as capsules,cachets or tablets, each containing a predetermined amount of the activeingredient; as a powder or granules; as a solution or a suspension in anaqueous liquid or a non-aqueous liquid; or as an oil-in-water liquidemulsion or a water-in-oil liquid emulsion. The active ingredient mayalso be presented as a bolus, electuary or paste.

Solutions or suspensions of the compound of the invention suitable fororal administration may also contain excipients e.g.N,N-dimethylacetamide, dispersants e.g. polysorbate 80, surfactants, andsolubilisers, e.g. polyethylene glycol, Phosal 50 PG (which consists ofphosphatidylcholine, soya-fatty acids, ethanol, mono/diglycerides,propylene glycol and ascorbyl palmitate). The formulations according topresent invention may also be in the form of emulsions, wherein acompound according to Formula (I) may be present in an aqueous oilemulsion. The oil may be any oil-like substance such as e.g. soy beanoil or safflower oil, medium chain triglycieride (MCT-oil) such as e.g.coconut oil, palm oil etc or combinations thereof.

Tablets may contain excipients such as microcrystalline cellulose,lactose (e.g. lactose monohydrate or lactose anyhydrous), sodiumcitrate, calcium carbonate, dibasic calcium phosphate and glycine,butylated hydroxytoluene (E321), crospovidone, hypromellose,disintegrants such as starch (preferably corn, potato or tapiocastarch), sodium starch glycollate, croscarmellose sodium, and certaincomplex silicates, and granulation binders such as polyvinylpyrrolidone,hydroxypropylmethylcellulose (HPMC), hydroxy-propylcellulose (HPC),macrogol 8000, sucrose, gelatin and acacia. Additionally, lubricatingagents such as magnesium stearate, stearic acid, glyceryl behenate andtalc may be included.

A tablet may be made by compression or moulding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder (e.g. povidone, gelatin, hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (e.g. sodium starchglycolate, crosslinked povidone, cross-linked sodium carboxymethylcellulose), surface-active or dispersing agent. Moulded tablets may bemade by moulding in a suitable machine a mixture of the powderedcompound moistened with an inert liquid diluent. The tablets mayoptionally be coated or scored and may be formulated so as to provideslow or controlled release of the active ingredient therein using, forexample, hydroxypropylmethylcellulose in varying proportions to providedesired release profile.

Solid compositions of a similar type may also be employed as fillers ingelatin capsules. Preferred excipients in this regard include lactose,starch, a cellulose, milk sugar or high molecular weight polyethyleneglycols. For aqueous suspensions and/or elixirs, the compounds of theinvention may be combined with various sweetening or flavouring agents,colouring matter or dyes, with emulsifying and/or suspending agents andwith diluents such as water, ethanol, propylene glycol and glycerin, andcombinations thereof.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavoured basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouth-washes comprising the active ingredient in asuitable liquid carrier.

Pharmaceutical compositions adapted for topical administration may beformulated as ointments, creams, suspensions, lotions, powders,solutions, pastes, gels, impregnated dressings, sprays, aerosols oroils, transdermal devices, dusting powders, and the like. Thesecompositions may be prepared via conventional methods containing theactive agent. Thus, they may also comprise compatible conventionalcarriers and additives, such as preservatives, solvents to assist drugpenetration, emollient in creams or ointments and ethanol or oleylalcohol for lotions. Such carriers may be present as from about 1% up toabout 98% of the composition. More usually they will form up to about80% of the composition. As an illustration only, a cream or ointment isprepared by mixing sufficient quantities of hydrophilic material andwater, containing from about 5-10% by weight of the compound, insufficient quantities to produce a cream or ointment having the desiredconsistency.

Pharmaceutical compositions adapted for transdermal administration maybe presented as discrete patches intended to remain in intimate contactwith the epidermis of the recipient for a prolonged period of time. Forexample, the active agent may be delivered from the patch byiontophoresis.

For applications to external tissues, for example the mouth and skin,the compositions are preferably applied as a topical ointment or cream.When formulated in an ointment, the active agent may be employed witheither a paraffinic or a water-miscible ointment base.

Alternatively, the active agent may be formulated in a cream with anoil-in-water cream base or a water-in-oil base.

For parenteral administration, fluid unit dosage forms are preparedutilizing the active ingredient and a sterile vehicle, for example butwithout limitation water, alcohols, polyols, glycerine and vegetableoils, water being preferred. The active ingredient, depending on thevehicle and concentration used, can be either colloidal, suspended ordissolved in the vehicle. In preparing solutions the active ingredientcan be dissolved in water for injection and filter sterilised beforefilling into a suitable vial or ampoule and sealing.

Advantageously, agents such as local anaesthetics, preservatives andbuffering agents can be dissolved in the vehicle. To enhance thestability, the composition can be frozen after filling into the vial andthe water removed under vacuum. The dry lyophilized powder is thensealed in the vial and an accompanying vial of water for injection maybe supplied to reconstitute the liquid prior to use.

Pharmaceutical compositions of the present invention suitable forinjectable use include sterile aqueous solutions or dispersions.Furthermore, the compositions can be in the form of sterile powders forthe extemporaneous preparation of such sterile injectable solutions ordispersions. In all cases, the final injectable form must be sterile andmust be effectively fluid for easy syringability.

Parenteral suspensions are prepared in substantially the same manner assolutions, except that the active ingredient is suspended in the vehicleinstead of being dissolved and sterilization cannot be accomplished byfiltration. The active ingredient can be sterilised by exposure toethylene oxide before suspending in the sterile vehicle. Advantageously,a surfactant or wetting agent is included in the composition tofacilitate uniform distribution of the active ingredient.

It should be understood that in addition to the ingredients particularlymentioned above the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example those suitable for oral administration mayinclude flavouring agents. A person skilled in the art will know how tochoose a suitable formulation and how to prepare it (see eg Remington'sPharmaceutical Sciences 18 Ed. or later). A person skilled in the artwill also know how to choose a suitable administration route and dosage.

It will be recognized by one of skill in the art that the optimalquantity and spacing of individual dosages of a compound of theinvention will be determined by the nature and extent of the conditionbeing treated, the form, route and site of administration, and the ageand condition of the particular subject being treated, and that aphysician will ultimately determine appropriate dosages to be used. Thisdosage may be repeated as often as appropriate. If side effects developthe amount and/or frequency of the dosage can be altered or reduced, inaccordance with normal clinical practice.

All % values mentioned herein are % w/w unless the context requiresotherwise.

Compounds of the invention all may be transformed in a biological matrixto liberate succinic acid, succinyl coenzyme A or canonical forms of thesame. They may do so as follows.

Where R′, R″ or R′″ is a compound of formula (II) the acyl groupincluding R₂ may be cleaved by a suitable enzyme, preferably anesterase. This liberates an hydroxymethyl ester, an aminomethyl ester ora thiolmethyl ester which could spontaneous covert to a carbonyl, imineor thiocarbonyl group and a free carboxylic acid. By way of example informula (I) where A is OR′ with R′ being formula (II) and B is H and Zis —CH₂CH₂—.

When B is —SR′″ a thiol group is released. This is regarded asespecially advantageous as the thiol group has reductive properties.Many diseases have an unwanted oxidative stress component, which maylead to damage to cell structure and cell function. Accordingly, releaseof a component which can act as an anti-oxidant and scavenge freeradicals or reduce oxygen-reactive species is expected to give extrabenefit in medical or cosmetic use.

Where R′, R″ or R′″ is a compound of formula (V) the substituent ongroup R₁₀ may be removed by the action of a suitable enzyme or viachemical hydrolysis in vivo. By way of example in formula (I) where A isOR′ with R′ being formula (V) and B is H and Z is —CH₂CH₂—, X is O andR8 is H, R9 is Me and R10 is O-acetyl.

Where R′, R″ or R′″ is a compound of formula (VII) the group may beremoved by the action of a suitable enzyme or via chemical hydrolysis invivo to liberate succinic acid. By way of example in formula (I) where Ais SR with R being formula (VII) and B is OH and Z is —CH₂CH₂—, X₅ isCO₂H and R₁ is Et:

Alternatively for compounds of formula VII the entity in itself may betaken directly into the Krebs cycle in the place of succinyl-CoA.

Where formula (I) is

the compound may hydrolyse to give a compound according to the schemebelow and when X₄ is —COOH.

Other Aspects of the Invention

The present invention also provides a combination (for example for thetreatment of mitochondrial dysfunction) of a compound of formula (I) ora pharmaceutically acceptable form thereof as hereinbefore defined andone or more agents independently selected from:

-   -   Quinone derivatives, e.g. Ubiquinone, Idebenone, MitoQ    -   Vitamins e.g. Tocopherols, Tocotrienols and Trolox (Vitamin E),        Ascorbate (C), Thiamine (B1), Riboflavin (B2), Nicotinamide        (B3), Menadione (K3),    -   Antioxidants in addition to vitamins e.g. TPP-compounds (MitoQ),        Sk-compounds, Epicatechin, Catechin, Lipoic acid, Uric acid,        Melatonin    -   Dichloroacetate    -   Methylene blue    -   I-arginine    -   Szeto-Schiller peptides    -   Creatine    -   Benzodiazepines    -   Modulators of PGC-1α    -   Ketogenic diet

One other aspect of the invention is that any of the compounds asdisclosed herein may be administered together with any other compoundssuch as e.g. sodium bicarbonate (as a bolus (e.g. 1 mEq/kg) followed bya continuous infusion.) as a concomitant medication to the compounds asdisclosed herein.

Lactic Acidosis or Drug-Induced Side-Effects Due to Complex I-RelatedImpairment of Mitochondrial Oxidative Phosphorylation

The present invention also relates to the prevention or treatment oflactic acidosis and of mitochondrial-related drug-induced side effects.In particular the compounds according to the invention are used in theprevention or treatment of a mitochondrial-related drug-induced sideeffects at or up-stream of Complex I, or expressed otherwise, theinvention provides according to the invention for the prevention ortreatment of drug-induced direct inhibition of Complex I or of anydrug-induced effect that limits the supply of NADH to Complex I (suchas, but not limited to, effects on Krebs cycle, glycolysis,beta-oxidation, pyruvate metabolism and even drugs that effects thetransport or levels of glucose or other complex I related substrates).

Mitochondrial toxicity induced by drugs may be a part of the desiredtherapeutic effect (e.g. mitochondrial toxicity induced by cancerdrugs), but in most case mitochondrial toxicity induced by drugs is anunwanted effect. Mitochondrial toxicity can markedly increase glycolysisto compensate for cellular loss of mitochondrial ATP formation byoxidative phosphorylation. This can result in increased lactate plasmalevels, which if excessive results in lactic acidosis, which can belethal. Type A lactic acidosis is primarily associated with tissuehypoxia, whereas type B aerobic lactic acidosis is associated withdrugs, toxin or systemic disorders such as liver diseases, diabetes,cancer and inborn errors of metabolism (e.g. mitochondrial geneticdefects).

Many known drug substances negatively influence mitochondrialrespiration (e.g. antipsychotics, local anaesthetics and anti-diabetics)and, accordingly, there is a need to identify or develop means thateither can be used to circumvent or alleviate the negative mitochondrialeffects induced by the use of such a drug substance.

The present invention provides compounds for use in the prevention ortreatment of lactic acidosis and of mitochondrial-related drug-inducedside effects. In particular the succinate prodrugs are used in theprevention or treatment of a mitochondrial-related drug-induced sideeffects at or up-stream of Complex I, or expressed otherwise, theinvention provides succinate prodrugs for the prevention or treatment ofdrug-induced direct inhibition of Complex I or of any drug-inducedeffect that limits the supply of NADH to Complex I (such as, but notlimited to, effects on Krebs cycle, glycolysis, beta-oxidation, pyruvatemetabolism and even drugs that effects the transport or levels ofglucose or other Complex I related substrates).

As mentioned above, increased lactate plasma levels are often observedin patients treated with drugs that may have mitochondrial-related sideeffects. The present invention is based on experimental results showingthat metformin (first-line treatment for type 2 diabetes and which hasbeen associated with lactic acidosis as a rare side-effect) inhibitsmitochondrial function of human peripheral blood cells at Complex I in atime- and dose-dependent fashion at concentrations relevant formetformin intoxication. Metformin further causes a significant increasein lactate production by intact platelets over time. The use of thecompounds according to the invention significantly reduced lactateproduction in metformin-exposed intact platelets. Exogenously appliedsuccinate, the substrate itself, did not reduce the metformin-inducedproduction of lactate.

In another study, the production of lactate was observed over severalhours in rotenone-inhibited platelets (i.e. a condition where thefunction of complex I is impaired). The use of the compounds accordingto the invention (but not succinate) attenuated the rotenone-inducedlactate production of intact human platelets. Respirometric experimentswere repeated in human fibroblasts and human heart muscle fibres, andconfirmed the findings seen in blood cells.

Accordingly, the invention provides compounds according to Formula (I)for use in the prevention of treatment of lactic acidosis. However, asthe results reported herein are based on lactic acidosis related todirect inhibition of Complex I or associated with a defect at orup-stream of Complex I, it is contemplated that the compounds accordingto the invention are suitable for use in the prevention or treatment ofa mitochondrial-related drug-induced side-effects at or up-stream ofComplex I. The compounds according to the invention would alsocounteract drug effects disrupting metabolism upstream of complex I(indirect inhibition of Complex I, which would encompass any drug effectthat limits the supply of NADH to Complex I, e.g. effects on Krebscycle, glycolysis, beta-oxidation, pyruvate metabolism and even drugsthat affect the levels of glucose or other complex I relatedsubstrates).

It is contemplated that the compounds according to the invention alsocan be used in industrial applications, e.g. in vitro to reduce orinhibit formation of lactate or to increase the ATP-availability ofcommercial or industrial cell lines. Examples include the use in cellculture, in organ preservation, etc.

The compounds according to the invention are used in the treatment orprevention of drug-induced mitochondrial-related side-effects or toincrease or restore cellular levels of energy (ATP), in the treatment.Especially, they are used in the treatment or prevention of direct orindirect drug-induced Complex I mitochondrial-related side-effects. Inparticular, they are used in the treatment or prevention of lacticacidosis, such as lactic acidosis induced by a drug substance.

The invention also relates to a combination of a compound of Formula (I)and a drug substance that may induce a mitochondrial-relatedside-effect, in particular a side-effect that is caused by direct orindirect impairment of Complex I by the drug substance. Such combinationcan be used as prophylactic prevention of a mitochondrial-relatedside-effect or, in case the side-effect appears, in alleviating and/ortreating the mitochondrial-related side effect.

It is contemplated that compounds as described below will be effectivein treatment or prevention of drug-induced side-effects, in particularin side-effects related to direct or indirect inhibition of Complex I.

Drug substances that are known to give rise in Complex I defects,malfunction or impairment and/or are known to have lactic acidosis asside-effect are:

Analgesics including acetaminophen, capsaicin

Antianginals including amiodarone, perhexiline

Antibiotics including linezolid, trovafloxacin, gentamycin

Anticancer drugs including quinones including mitomycin C, adriamycin

Anti-convulsant drugs including valproic acid

Anti-diabetics including metformin, phenformin, butylbiguanide,troglitazone and rosiglitazone, pioglitazone

Anti-Hepatitis B including fialuridine

Antihistamines

Anti-Parkinson including tolcapone

Anti-psycotics Risperidone,

Anti-schizoprenia zotepine, clozapine

Antiseptics, quaternary ammonium compounds (QAC)

Anti-tuberculosis including isoniazid

Fibrates including clofibrate, ciprofibrate, simvastatin

Hypnotics including Propofol

Immunosupressive disease-modifying antirheumatic drug (DMARD)Leflunomide

Local anaesthetics including bupivacaine, diclofenac, indomethacin, andlidocaine

Muscle relaxant including dantrolene

Neuroleptics including antipsycotic neuroleptics like chlorpromazine,fluphenazine and haloperidol

NRTI (Nucleotide reverse Transcriptase Inhibitors) including efavirenz,tenofovir, emtricitabine, zidovudine, lamivudine, rilpivirine, abacavir,didanosine

NSAIDs including nimesulfide, mefenamic acid, sulindac

Barbituric acids.

Other drug substances that are known to have lactic acidosis asside-effects include beta2-agonists, epinephrine, theophylline or otherherbicides. Alcohols and cocaine can also result in lactic acidosis.

Moreover, it is contemplated that the compounds of the invention alsomay be effective in the treatment or prevention of lactic acidosis evenif it is not related to a Complex I defect.

Combination of Drugs and Compounds of the Invention

The present invention also relates to a combination of a drug substanceand a compound of the invention for use in the treatment and/orprevention of a drug-induced side-effect selected from lactic acidosisand side-effect related to a Complex I defect, inhibition ormalfunction, wherein

i) the drug substance is used for treatment of a disease for which thedrug substance is indicated, and

ii) the compound of the invention is used for prevention or alleviationof the side effects induced or inducible by the drug substance, whereinthe side-effects are selected from lactic acidosis and side-effectsrelated to a Complex I defect, inhibition or malfunction.

Any combination of such a drug substance with any compound of theinvention is within the scope of the present invention. Accordingly,based on the disclosure herein a person skilled in the art willunderstand that the gist of the invention is the findings of thevaluable properties of compounds of the invention to avoid or reduce theside-effects described herein. Thus, the potential use of compounds ofthe invention capable of entering cells and deliver succinate andpossibly other active moeties in combination with any drug substancethat has or potentially have the side-effects described herein isevident from the present disclosure.

The invention further relates to

i) a composition comprising a drug substance and a compound of theinvention, wherein the drug substance has a potential drug-inducedside-effect selected from lactic acidosis and side-effects related to aComplex I defect, inhibition or malfunction,

ii) a composition as described above under i), wherein the compound ofthe invention is used for prevention or alleviation of side effectsinduced or inducible by the drug substance, wherein the side-effects areselected from lactic acidosis and side-effects related to a Complex Idefect, inhibition or malfunction.

The composition may be in the form of two separate packages:

A first package containing the drug substance or a compositioncomprising the drug substance and

a second package containing the compound of the invention or acomposition comprising the compound of the invention. The compositionmay also be a single composition comprising both the drug substance andthe compound of the invention.

In the event that the composition comprises two separate packages, thedrug substance and the compound of the invention may be administered bydifferent administration routes (e.g. drug substance via oraladministration and compound of the invention by parenteral or mucosaladministration) and/or they may be administered essentially at the sametime or the drug substance may be administered before the compound ofthe invention or vice versa.

Kits

The invention also provides a kit comprising

i) a first container comprising a drug substance, which has a potentialdrug-induced side-effect selected from lactic acidosis and side-effectsrelated to a Complex I defect, inhibition or malfunction, and

ii) a second container comprising a compound of the invention, which hasthe potential for prevention or alleviation of the side effects inducedor inducible by the drug substance, wherein the side-effects areselected from lactic acidosis and side-effects related to a Complex Idefect, inhibition or malfunction.

Method for Treatment/Prevention of Side-Effects

The invention also relates to a method for treating a subject sufferingfrom a drug-induced side-effect selected from lactic acidosis andside-effect related to a Complex I defect, inhibition or malfunction,the method comprises administering an effective amount of a compound ofthe invention to the subject, and to a method for preventing oralleviating a drug-induced side-effect selected from lactic acidosis andside-effect related to a Complex I defect, inhibition or malfunction ina subject, who is suffering from a disease that is treated with a drugsubstance, which potentially induce a side-effect selected from lacticacidosis and side-effect related to a Complex I defect, inhibition ormalfunction, the method comprises administering an effective amount of acompound of the invention to the subject before, during or aftertreatment with said drug substance.

Metformin

Metformin is an anti-diabetic drug belonging to the class of biguanides.It's the first line treatment for type 2 diabetes, which accounts foraround 90% of diabetes cases in the USA. The anti-diabetic effect hasbeen attributed to decreasing hepatic glucose production, increasing thebiological effect of insulin through increased glucose uptake inperipheral tissues and decreasing uptake of glucose in the intestine,but the exact mechanisms of action have not been completely elucidated.Despite its advantages over other anti-diabetics it has been related torare cases of lactic acidosis (LA) as side effect). LA is defined as anincreased anion gap, an arterial blood lactate level above 5 mM and apH≤7.35. Although the precise pathogenesis of metformin-associated LA isstill not completely revealed, an inhibition of gluconeogenesis andresulting accumulation of gluconeogenic precursors, such as alanine,pyruvate and lactate, has been suggested. Others, however, propose aninterference of the drug with mitochondrial function being the keyfactor for both the primary therapeutic, glucose-lowering effect as wellas for the development of metformin-associated LA). As a consequence ofmitochondrial inhibition, the cell would partly shift from aerobic toanaerobic metabolism, promoting glycolysis with resulting elevatedlactate levels. Phenformin, another antidiabetic agent of the same drugclass as metformin, has been withdrawn from the market in most countriesdue to a high incidence of LA (4 cases per 10000 treatmentyears). Incomparison, the incidence of LA for metformin is about a tenth of thatfor phenformin, and it is therefore considered a rather safe therapeuticagent. Metformin-associated LA is seen mostly in patients who haveadditional predisposing conditions affecting the cardiovascular system,liver or kidneys. Under these conditions, the drug clearance from thebody is impaired which, if not detected in time, results in escalatingblood concentrations of metformin. Since the use of metformin isexpected to rise due to increasing prevalence of type 2 diabetes, theresearch on metformin-induced mitochondrial toxicity and LA becomes acurrent and urgent issue. Research on the mitochondrial toxicity ofmetformin reports inconsistent results. Kane et al. (2010) did notdetect inhibition of basal respiration and maximal respiratorycapacities by metformin in vivo in skeletal muscle from rats and neitherdid in muscle biopsies of metformin-treated type 2 diabetes patients. Incontrast, others have described toxic effects of metformin andphenformin on mitochondria and its association with LA in animaltissues. Data on human tissue are scarce, especially ex vivo or in vivo.Most human data on metformin and LA are based on retrospective studiesdue to the difficulty of obtaining human tissue samples. Protti et al(2010), however, reported decreased systemic oxygen consumption inpatients with biguanide-associated LA and both Protti et al (2012b) andLarsen et al. (2012) described mitochondrial dysfunction in vitro inresponse to metformin exposure at 10 mM in human skeletal muscle andplatelets, respectively. Protti et al. (2012b further reported onincreased lactate release in human platelets in response to metforminexposure at 1 mM. Although metformin is not found at this concentrationat therapeutic conditions, it has been shown to approach these levels inthe blood during intoxication and it is known to accumulate 7 to 10-foldin the gastrointestinal tract, kidney, liver, salivary glands, lung,spleen and muscle as compared to plasma.

In the study reported herein the aim was to assess mitochondrialtoxicity of metformin and phenformin in human blood cells usinghigh-resolution respirometry. Phenformin was included to compareactivity of the two similarly structured drugs and to study the relationbetween mitochondrial toxicity and the incidence of LA described inhuman patients. In order to investigate membrane permeability and thespecific target of toxicity of these biguanides, a model for testingdrug toxicity was applied using both intact and permeabilized bloodcells with sequential additions of respiratory complex-specificsubstrates and inhibitors.

Other aspects appear from the appended claims. All details andparticulars apply mutatis mutandis to these aspects.

Definitions

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. at least one) of the grammatical objects of the article.By way of example “an analogue” means one analogue or more than oneanalogue.

As used herein the terms “cell permeable succinates”, “compound(s) ofthe invention”, “cell-permeable succinate derivatives” and “cellpermeable precursors of succinate” are used interchangeably and refer tocompounds of formula (I).

As used herein, the term “bioavailability” refers to the degree to whichor rate at which a drug or other substance is absorbed or becomesavailable at the site of biological activity after administration. Thisproperty is dependent upon a number of factors including the solubilityof the compound, rate of absorption in the gut, the extent of proteinbinding and metabolism etc. Various tests for bioavailability that wouldbe familiar to a person of skill in the art are described herein (seealso Trepanier et al, 1998, Gallant-Haidner et al, 2000).

As used herein the terms “impairment”, inhibition”, “defect” used inrelation to Complex I of the respiratory chain is intended to denotethat a given drug substance have negative effect on Complex I or onmitochondrial metabolism upstream of Complex I, which could encompassany drug effect that limits the supply of NADH to Complex I, e.g.effects on Krebs cycle, glycolysis, beta-oxidation, pyruvate metabolismand even drugs that effect the transport or levels of glucose or othercomplex I-related substrates). As described herein, an excess of lactatein a subject is often an indication of a negative effect on aerobicrespiration including Complex I.

As used herein the term “side-effect” used in relation to the functionof Complex I of the respiratory chain may be a side-effect relating tolactic acidosis or it may be a side-effect relating to idiosyncraticdrug organ toxicity e.g. hepatotoxicity, neurotoxicity, cardiotoxicity,renal toxicity and muscle toxicity encompassing, but not limited to,e.g. ophthalmoplegia, myopathy, sensorineural hearing impairment,seizures, stroke, stroke-like events, ataxia, ptosis, cognitiveimpairment, altered states of consciousness, neuropathic pain,polyneuropathy, neuropathic gastrointestinal problems (gastroesophagealreflux, constipation, bowel pseudo-obstruction), proximal renal tubulardysfunction, cardiac conduction defects (heart blocks), cardiomyopathy,hypoglycemia, gluconeogenic defects, nonalcoholic liver failure, opticneuropathy, visual loss, diabetes and exocrine pancreatic failure,fatigue, respiratory problems including intermittent air hunger.

As used herein the term “drug-induced” in relation to the term“side-effect” is to be understood in a broad sense. Thus, not only doesit include drug substances, but also other substances that may lead tounwanted presence of lactate. Examples are herbicides, toxic mushrooms,berries etc.

The pharmaceutically acceptable salts of the compound of the inventioninclude conventional salts formed from pharmaceutically acceptableinorganic or organic acids or bases as well as quaternary ammonium acidaddition salts. More specific examples of suitable acid salts includehydrochloric, hydrobromic, sulfuric, phosphoric, nitric, perchloric,fumaric, acetic, propionic, succinic, glycolic, formic, lactic, maleic,tartaric, citric, palmoic, malonic, hydroxymaleic, phenylacetic,glutamic, benzoic, salicylic, fumaric, toluenesulfonic, methanesulfonic,naphthalene-2-sulfonic, benzenesulfonic hydroxynaphthoic, hydroiodic,malic, steroic, tannic and the like. Other acids such as oxalic, whilenot in themselves pharmaceutically acceptable, may be useful in thepreparation of salts useful as intermediates in obtaining the compoundsof the invention and their pharmaceutically acceptable salts. Morespecific examples of suitable basic salts include sodium, lithium,potassium, magnesium, aluminium, calcium, zinc,N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, Nmethylglucamine and procaine salts.

As used herein the term “alkyl” refers to any straight or branched chaincomposed of only sp3 carbon atoms, fully saturated with hydrogen atomssuch as e.g. —C_(n)H_(2n+1) for straight chain alkyls, wherein n can bein the range of 1 and 10 such as e.g. methyl, ethyl, propyl, isopropyl,n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, neopentyl,isopentyl, hexyl, isohexyl, heptyl, octyl, nonyl or decyl. The alkyl asused herein may be further substituted.

As used herein the term “cycloalkyl” refers to a cyclic/ring structuredcarbon chains having the general formula of —C_(n)H_(2n−1) where n isbetween 3-10, such as e.g. cyclopropyl, cyclobytyl, cyclopentyl,cyclohexyl, cycloheptyl or cyclooctyl, bicycle[3.2.1]octyl,spiro[4,5]decyl, norpinyl, norbonyl, norcapryl, adamantly and the like.

As used herein, the term “alkene” refers to a straight or branched chaincomposed of carbon and hydrogen atoms wherein at least two carbon atomsare connected by a double bond such as e.g. C₂₋₁₀ alkenyl unsaturatedhydrocarbon chain having from two to ten carbon atoms and at least onedouble bond. C₂₋₆ alkenyl groups include, but are not limited to, vinyl,1-propenyl, allyl, iso-propenyl, n-butenyl, n-pentenyl, n-hexenyl andthe like.

The term “C₁₋₁₀ alkoxy” in the present context designates a group—O—C-₁₋₆ alkyl used alone or in combination, wherein C₁₋₁₀ alkyl is asdefined above. Examples of linear alkoxy groups are methoxy, ethoxy,propoxy, butoxy, pentoxy and hexoxy. Examples of branched alkoxy areiso-propoxy, sec-butoxy, tert-butoxy, iso-pentoxy and isohexoxy.Examples of cyclic alkoxy are cyclopropyloxy, cyclobutyloxy,cyclopentyloxy and cyclohexyloxy.

The term “C₃₋₇ heterocycloalkyl” as used herein denotes a radical of atotally saturated heterocycle like a cyclic hydrocarbon containing oneor more heteroatoms selected from nitrogen, oxygen and sulphurindependently in the cycle. Examples of heterocycles include, but arenot limited to, pyrrolidine (1-pyrrolidine, 2-pyrrolidine,3-pyrrolidine, 4-pyrrolidine, 5-pyrrolidine), pyrazolidine(1-pyrazolidine, 2-pyrazolidine, 3-pyrazolidine, 4-pyrazolidine,5-pyrazolidine), imidazolidine (1-imidazolidine, 2-imidazolidine,3-imidazolidine, 4-imidazolidine, 5-imidazolidine), thiazolidine(2-thiazolidine, 3-thiazolidine, 4-thiazolidine, 5-thiazolidine),piperidine (1-piperidine, 2-piperidine, 3-piperidine, 4-piperidine,5-piperidine, 6-piperidine), piperazine (1-piperazine, 2-piperazine,3-piperazine, 4-piperazine, 5-piperazine, 6-piperazine), morpholine(2-morpholine, 3-morpholine, 4-morpholine, 5-morpholine, 6-morpholine),thiomorpholine (2-thiomorpholine, 3-thiomorpholine, 4-thiomorpholine,5-thiomorpholine, 6-thiomorpholine), 1,2-oxathiolane(3-(1,2-oxathiolane), 4-(1,2-oxathiolane), 5-(1,2-oxathiolane)),1,3-dioxolane (2-(1,3-dioxolane), 3-(1,3-dioxolane), 4-(1,3-dioxolane)),tetrahydropyrane (2-tetrahydropyrane, 3-tetrahydropyrane,4-tetrahydropyrane, 5-tetrahydropyrane, 6-tetrahydropyrane),hexahydropyradizine, (1-(hexahydropyradizine), 2-(hexahydropyradizine),3-(hexahydropyradizine), 4-(hexahydropyradizine),5-(hexahydropyradizine), 6-(hexahydropyradizine)).

The term “C₁₋₁₀alkyl-C₃₋₁₀ cycloalkyl” as used herein refers to acycloalkyl group as defined above attached through an alkyl group asdefined above having the indicated number of carbon atoms.

The term “C₁₋₁₀ alkyl-C₃₋₇ heterocycloalkyl” as used herein refers to aheterocycloalkyl group as defined above attached through an alkyl groupas defined above having the indicated number of carbon atoms.

The term “aryl” as used herein is intended to include carbocyclicaromatic ring systems. Aryl is also intended to include the partiallyhydrogenated derivatives of the carbocyclic systems enumerated below.

The term “heteroaryl” as used herein includes heterocyclic unsaturatedring systems containing one or more heteroatoms selected among nitrogen,oxygen and sulphur, such as furyl, thienyl, pyrrolyl, and is alsointended to include the partially hydrogenated derivatives of theheterocyclic systems enumerated below.

The terms “aryl” and “heteroaryl” as used herein refers to an aryl,which can be optionally unsubstituted or mono-, di- or tri substituted,or a heteroaryl, which can be optionally unsubstituted or mono-, di- ortri substituted. Examples of “aryl” and “heteroaryl” include, but arenot limited to, phenyl, biphenyl, indenyl, naphthyl (1-naphthyl,2-naphthyl), N-hydroxytetrazolyl, N-hydroxytriazolyl,N-hydroxyimidazolyl, anthracenyl (1-anthracenyl, 2-anthracenyl,3-anthracenyl), phenanthrenyl, fluorenyl, pentalenyl, azulenyl,biphenylenyl, thiophenyl (1-thienyl, 2-thienyl), furyl (1-furyl,2-furyl), furanyl, thiophenyl, isoxazolyl, isothiazolyl,1,2,3-triazolyl, 1,2,4-triazolyl, pyranyl, pyridazinyl, pyrazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl, 1,3,5-triazinyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1 ,2,5-oxadiazolyl, 1,3,4-oxadiazolyl,1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl,1,3,4-thiadiazolyl, tetrazolyl, thiadiazinyl, indolyl, isoindolyl,benzofuranyl, benzothiophenyl (thianaphthenyl), indolyl, oxadiazolyl,isoxazolyl, quinazolinyl, fluorenyl, xanthenyl, isoindanyl, benzhydryl,acridinyl, benzisoxazolyl, purinyl, quinazolinyl, quinolizinyl,quinolinyl, isoquinolinyl, quinoxalinyl, naphthyridinyl, phteridinyl,azepinyl, diazepinyl, pyrrolyl (2-pyrrolyl), pyrazolyl (3-pyrazolyl),5-thiophene-2-yl-2H-pyrazol-3-yl, imidazolyl (1-imidazolyl,2-imidazolyl, 4-imidazolyl, 5-imidazolyl), triazolyl(1,2,3-triazol-1-yl, 1,2,3-triazol-2-yl, 1,2,3-triazol-4-yl,1,2,4-triazol-3-yl), oxazolyl (2-oxazolyl, 4-oxazolyl, 5-oxazolyl),thiazolyl (2-thiazolyl, 4-thiazolyl, 5-thiazolyl), pyridyl (2-pyridyl,3-pyridyl, 4-pyridyl), pyrimidinyl (2-pyrimidinyl, 4-pyrimidinyl,5-pyrimidinyl, 6-pyrimidinyl), pyrazinyl, pyridazinyl (3-pyridazinyl,4-pyridazinyl, 5-pyridazinyl), isoquinolyl (1-isoquinolyl,3-isoquinolyl, 4-isoquinolyl, 5-isoquinolyl, 6-isoquinolyl,7-isoquinolyl, 8-isoquinolyl), quinolyl (2-quinolyl, 3-quinolyl,4-quinolyl, 5-quinolyl, 6-quinolyl, 7-quinolyl, 8-quinolyl),benzo[b]furanyl (2-benzo[b]furanyl, 3-benzo[b]furanyl,4-benzo[b]furanyl, 5-benzo[b]furanyl, 6-benzo[b]furanyl,7-benzo[b]furanyl), 2,3-dihydro-benzo[b]furanyl(2-(2,3-dihydro-benzo[b]furanyl), 3-(2,3-dihydro-benzo[b]furanyl),4-(2,3-dihydrobenzo[b]furanyl), 5-(2,3-dihydro-benzo[b]furanyl),6-(2,3-dihydro-benzo[b]furanyl), 7-(2,3-dihydro-benzo[b]furanyl)),benzo[b]thiophenyl (2-benzo[b]thiophenyl, 3-benzo[b]thiophenyl,4-benzo[b]thiophenyl, 5-benzo[b]thiophenyl, 6-benzo[b]thiophenyl,7-benzo[b]thiophenyl), 2,3-dihydro-benzo[b]thiophenyl(2-(2,3-dihydro-benzo[b]thiophenyl), 3-(2,3-dihydro-benzo[b]thiophenyl),4-(2,3-dihydro-benzo[b]thiophenyl), 5-(2,3-dihydro-benzo[b]thiophenyl),6-(2,3-dihydro-benzo[b]thiophenyl), 7-(2,3-dihydrobenzo[b]thiophenyl)),indolyl (1-indolyl, 2-indolyl, 3-indolyl, 4-indolyl, 5-indolyl,6-indolyl, 7-indolyl), indazolyl (1-indazolyl, 2-indazolyl, 3-indazolyl,4-indazolyl, 5-indazolyl, 6-indazolyl, 7-indazolyl), benzimidazolyl,(1-benzimidazolyl, 2-benzimidazolyl, 4-benzimidazolyl, 5-benzimidazolyl,6-benzimidazolyl, 7-benzimidazolyl, 8-benzimidazolyl), benzoxazolyl(1-benzoxazolyl, 2-benzoxazolyl), benzothiazolyl (1-benzothiazolyl,2-benzothiazolyl, 4-benzothiazolyl, 5-benzothiazolyl, 6-benzothiazolyl,7-benzothiazolyl), carbazolyl (1-carbazolyl, 2-carbazolyl, 3-carbazolyl,4-carbazolyl). Non-limiting examples of partially hydrogenatedderivatives are 1,2,3,4-tetrahydronaphthyl, 1,4-dihydronaphthyl,pyrrolinyl, pyrazolinyl, indolinyl, oxazolidinyl, oxazolinyl, oxazepinyland the like.

As used herein the term “acyl” refers to a carbonyl group —C(═O) Rwherein the R group is any of the above defined groups. Specificexamples are formyl, acetyl, propionyl, butyryl, pentanoyl, hexanoyl,heptanoyl, octanoyl, nonanoyl, decanoyl, benzoyl and the likes.

“Optionally substituted” as applied to any group means that the saidgroup may, if desired, be substituted with one or more substituents,which may be the same or different. ‘Optionally substituted alkyl’includes both ‘alkyl’ and ‘substituted alkyl’.

Examples of suitable substituents for “substituted” and “optionallysubstituted” moieties include halo (fluoro, chloro, bromo or iodo), C₁₋₆alkyl, C₃₋₆ cycloalkyl, hydroxy, C₁₋₆ alkoxy, cyano, amino, nitro, C₁₋₆alkylamino, C₂₋₆ alkenylamino, alkylamino, C₁₋₆ acylamino, acylamino,C₁₋₆ aryl, C₁₋₆ arylamino, C₁₋₆ aroylamino, benzylamino, C₁₋₆ arylamido,carboxy, C₁₋₆ alkoxycarbonyl or (C₁₋₆ aryl)(C₁₋₁₀ alkoxy)carbonyl,carbamoyl, mono-C₁₋₆ carbamoyl, carbamoyl or any of the above in which ahydrocarbyl moiety is itself substituted by halo, cyano, hydroxy, C₁₋₂alkoxy, amino, nitro, carbamoyl, carboxy or C₁₋₂ alkoxycarbonyl. Ingroups containing an oxygen atom such as hydroxy and alkoxy, the oxygenatom can be replaced with sulphur to make groups such as thio (SH) andthio-alkyl (S-alkyl). Optional substituents therefore include groupssuch as S-methyl. In thio-alkyl groups, the sulphur atom may be furtheroxidised to make a sulfoxide or sulfone, and thus optional substituentstherefore includes groups such as S(O)-alkyl and S(O)₂-alkyl.

Substitution may take the form of double bonds, and may includeheteroatoms. Thus an alkyl group with a carbonyl (C═) instead of a CH₂can be considered a substituted alkyl group.

Substituted groups thus include for example CFH₂, CF₂H, CF₃, CH₂NH₂,CH₂OH, CH₂CN, CH₂SCH₃, CH₂OCH₃, OMe, OEt, Me, Et, —OCH₂O—, CO₂Me,C(O)Me, i-Pr, SCF₃, SO₂Me, NMe₂, CONH₂, CONMe₂etc. In the case of arylgroups, the substitutions may be in the form of rings from adjacentcarbon atoms in the aryl ring, for example cyclic acetals such asO—CH₂—O.

The invention is illustrated in the following figures:

FIG. 1. Schematic figure of evaluation assay for enhancement ofmitochondrial energy producing function in complex I inhibited cells.Protocol for evaluating the compounds according to the invention. In theassay, mitochondrial function in intact cells is repressed with therespiratory complex I inhibitor rotenone. Drug candidates are comparedwith endogenous (non cell-permeable) substrates before and afterpermeabilization of the plasma membrane to evaluate bioenergeticenhancement or inhibition.

FIG. 2. Schematic figure of assay for enhancement and inhibition ofmitochondrial energy producing function in intact cells. Protocol forevaluating the potency of compounds according to the invention. In theassay, mitochondrial activity is stimulated by uncoupling themitochondria with the protonophore FCCP. Drug candidates are titrated toobtain the level of maximum convergent (complex I- and complexII-derived) respiration. After rotenone addition, complex II-dependentstimulation is obtained. The complex III-inhibitor Antimycin is added toevaluate non mitochondrial oxygen consumption.

FIG. 3. Schematic figure of assay for prevention of lactate accumulationin cells exposed to a mitochondrial complex I inhibitor. Protocol forevaluating the potency of compounds according to the invention. In theassay, mitochondrial function in intact cells is repressed with therespiratory complex I inhibitor rotenone. As the cells shift toglycolysis lactate is accumulated in the medium. Drug candidates arecompared with endogenous (non cell-permeable) substrates and decreasedrate of lactate accumulation indicates restoration of mitochondrial ATPproduction.

FIG. 4. Figure of lactate accumulation in an acute metabolic crisismodel in pig.

Lactate accumulation in an acute metabolic crisis model in pig. In theanimal model, mitochondrial function is repressed by infusion of therespiratory complex I inhibitor rotenone. As the cells shift toglycolysis lactate is accumulated in the body. Mean arterial lactateconcentrations are demonstrated for rotenone and vehicle treated animalsat indicated infusion rates. Drug candidates are evaluated in rotenonetreated animals and decreased rate of lactate accumulation indicatesrestoration of mitochondrial ATP production.

FIGS. 5A-5C Effect of metformin on mitochondrial respiration inpermeabilized human peripheral blood mononuclear cells (PBMCs) andplatelets. (FIG. 5A) Representative traces of simultaneously measured O₂consumption of metformin—(1 mM, black trace) or vehicle-treated (H₂O,grey trace) permeabilized PBMCs assessed by applying sequentialadditions of indicated respiratory complex-specific substrates andinhibitors. The stabilization phase of the traces, disturbances due toreoxygenation of the chamber and complex IV substrate administrationhave been omitted (dashed lines). Boxes below traces state therespiratory complexes utilized for respiration during oxidation of thegiven substrates, complex I (CI), complex II (CII) or both (CI+II), aswell as the respiratory states at the indicated parts of the protocol.Respiratory rates at three different respiratory states and substratecombinations are illustrated for PBMCs (FIG. 5B) and platelets (FIG. 5C)for control (H₂O) and indicated concentrations of metformin: oxidativephosphorylation capacity supported by complex I substrates(OXPHOS_(CI)), complex II-dependent maximal flux through the electrontransport system (ETS_(CII)) Following titration of the protonophoreFCCP, and complex IV (CIV) capacity. Values are depicted as mean±SEM.*=P<0.05, **=P<0.01 and ***=P<0.001 using one-way ANOVA withHolm-Sidak's multiple comparison method, n=5. OXPHOS=oxidativephosphorylatation. ETS=electron transport system. ROX=residual oxygenconcentration.

FIG. 6 Dose-response comparison of the toxicity displayed by metforminand phenformin on mitochondrial respiratory capacity during oxidativephosphorylation supported by complex I-linked substrates (OXPHOS_(CI))in permeabilized human platelets. Rates of respiration are presented asmean±SEM and standard non-linear curve fitting was applied to obtainhalf maximal inhibitory concentration (IC₅₀) values for metformin andphenformin. *=P<0.05, **=P<0.01 and ***=P<0.001 compared to controlusing one-way ANOVA with Holm-Sidak's multiple comparison method, n=5.

FIGS. 7A-7B Time- and dose-dependent effects of metformin onmitochondrial respiration in intact human platelets. (FIG. 7A) Routinerespiration of platelets, i.e. respiration of the cells with theirendogenous substrate supply and ATP demand, was monitored during 60 minincubation of indicated concentrations of metformin or vehicle (H2O),which was followed by (FIG. 7B) maximal respiratory capacity induced bytitration of the protonophore FCCP to determine maximal flux through theelectron transport system (ETS) of the intact cells. Data are expressedas mean±SEM, n=5. *=P<0.05, **=P<0.01 and ***=P<0.001 using one-wayANOVA (b) and two-way ANOVA (a) with Holm-Sidak's post-hoc test.

FIG. 8 Effect of metformin and phenformin on lactate production and pHin suspensions of intact human platelets. Platelets were incubated inphosphate buffered saline containing glucose (10 mM) for 8 h with eithermetformin (10 mM, 1 mM), phenformin (0.5 mM), the complex I inhibitorrotenone (2 μM), or vehicle (DMSO, control). (Top) Lactate levels weredetermined every 2 h (n=5), and (bottom) pH was measured every 4 h(n=4). Data are expressed as mean±SEM. *=P<0.05, **=P<0.01 and***=P<0.001 using two-way ANOVA with Holm-Sidak's post-hoc test.

FIG. 9 Human intact thrombocytes (200·10⁶/ml) incubated in PBScontaining 10 mM glucose. (Left) Cells incubated with 10 mM metforminwere treated with either succinate or NV118 in consecutive additions of250 μM each 30 minutes. Prior to addition of NV118 at time 0 h, cellshave been incubated with just metformin or vehicle for 1 h to establishequal initial lactate levels (data not shown). Lactate concentrationswere sampled each 30 minutes. (Center) Lactate production was calculatedwith a non-linear fit regression and 95% confidence intervals for thetime lactate curves were calculated. Cells incubated with metformin hada significantly higher production of lactate than control, and succinateadditions did not change this. Lactate production was significantlydecreased when NV118 was added to the cells incubated with metformin.(Right) Lactate production induced by rotenone could similarly beattenuated by repeated additions of NV118.

FIG. 10 Human intact thrombocytes (200·10⁶/ml) incubated in PBScontaining 10 mM glucose. (Left) Cells incubated with 10 mM metforminwere treated with either succinate or NV189 in consecutive additions of250 μM each 30 minutes. Prior to addition of NV189 at time 0 h, cellshave been incubated with just metformin or vehicle for 1 h to establishequal initial lactate levels (data not shown). Lactate concentrationswere sampled each 30 minutes. (Center) Lactate production was calculatedwith a non-linear fit regression and 95% confidence intervals for thetime lactate curves were calculated. Cells incubated with metformin hada significantly higher production of lactate than control, and succinateadditions did not change this. Lactate production was significantlydecreased when NV189 was added to the cells incubated with metformin.(Right) Lactate production induced by rotenone could similarly beattenuated by repeated additions of NV189. When antimycin also wasadded, the effect of NV189 on complex 2 was abolished by antimycin'sinhibitory effect on complex III.

FIG. 11 Human intact thrombocytes (200·10⁶/ml) incubated in PBScontaining 10 mM glucose. (Left) Cells incubated with 10 mM metforminwere treated with either succinate or NV241 in consecutive additions of250 μM each 30 minutes. Prior to addition of NV241 at time 0 h, cellshave been incubated with just metformin or vehicle for 1 h to establishequal initial lactate levels (data not shown). Lactate concentrationswere sampled each 30 minutes. (Center) Lactate production was calculatedwith a non-linear fit regression and 95% confidence intervals for thetime lactate curves were calculated. Cells incubated with metformin hada significantly higher production of lactate than control, and succinateadditions did not change this. Lactate production was significantlydecreased when NV241 was added to the cells incubated with metformin.(Right) Lactate production induced by rotenone could similarly beattenuated by repeated additions of NV241.

FIGS. 12A-12B Thrombocytes (200·10⁶/ml) incubated in PBS containing 10mM of glucose with sampling of lactate concentrations every 30 minutes.(FIG. 12A) During 3 hour incubation, cells treated with either rotenone(2 μM) or its vehicle is monitored for change in lactate concentrationin media over time. Also, cells were incubated with rotenone togetherwith NV189 and cells with rotenone, NV189 and the complex Ill inhibitorantimycin (1 μg/mL) are monitored. Prior to addition of NV189 at time 0h, cells have been incubated with just rotenone or vehicle for 1 h toestablish equal initial lactate levels (data not shown). Rotenoneincrease the lactate production of the cells, but this is brought backto normal (same curve slope) by co-incubation with NV189 (in consecutiveadditions of 250 μM each 30 minutes). When antimycin also is present,NV189 cannot function at complex II level, and lactate production isagain increased to the same level as with only rotenone present. (FIG.12B) A similar rate of lactate production as with rotenone can beinduced by incubation with Metformin at 10 mM concentration.

EXPERIMENTAL

General Biology Methods

A person of skill in the art will be able to determine thepharmacokinetics and bioavailability of the compound of the inventionusing in vivo and in vitro methods known to a person of skill in theart, including but not limited to those described below and inGallant-Haidner et al, 2000 and Trepanier et al, 1998 and referencestherein. The bioavailability of a compound is determined by a number offactors, (e.g. water solubility, cell membrane permeability, the extentof protein binding and metabolism and stability) each of which may bedetermined by in vitro tests as described in the examples herein, itwill be appreciated by a person of skill in the art that an improvementin one or more of these factors will lead to an improvement in thebioavailability of a compound. Alternatively, the bioavailability of thecompound of the invention may be measured using in vivo methods asdescribed in more detail below, or in the examples herein.

In order to measure bioavailability in vivo, a compound may beadministered to a test animal (e.g. mouse or rat) both intraperitoneally(i.p.) or intravenously (i.v.) and orally (p.o.) and blood samples aretaken at regular intervals to examine how the plasma concentration ofthe drug varies over time. The time course of plasma concentration overtime can be used to calculate the absolute bioavailability of thecompound as a percentage using standard models. An example of a typicalprotocol is described below.

For example, mice or rats are dosed with 1 or 3 mg/kg of the compound ofthe invention i.v. or 1, 5 or 10 mg/kg of the compound of the inventionp.o. Blood samples are taken at 5 min, 15 min, 1 h, 4 h and 24 hintervals, and the concentration of the compound of the invention in thesample is determined via LCMS-MS. The time-course of plasma or wholeblood concentrations can then be used to derive key parameters such asthe area under the plasma or blood concentration-time curve (AUC—whichis directly proportional to the total amount of unchanged drug thatreaches the systemic circulation), the maximum (peak) plasma or blooddrug concentration, the time at which maximum plasma or blood drugconcentration occurs (peak time), additional factors which are used inthe accurate determination of bioavailability include: the compound'sterminal half-life, total body clearance, steady-state volume ofdistribution and F %. These parameters are then analysed bynon-compartmental or compartmental methods to give a calculatedpercentage bioavailability, for an example of this type of method seeGallant-Haidner et al, 2000 and Trepanier et al, 1998, and referencestherein.

The efficacy of the compound of the invention may be tested using one ormore of the methods described below:

I. Assays for Evaluating Enhancement and Inhibition of MitochondrialEnergy Producing Function in Intact Cells

High Resolution Respirometry—A—General Method

Measurement of mitochondrial respiration is performed in ahigh-resolution oxygraph (Oxygraph-2k, Oroboros Instruments, Innsbruck,Austria) at a constant temperature of 37° C. Isolated human platelets,white blood cells, fibroblasts, human heart muscle fibers or other celltypes containing live mitochondria are suspended in a 2 mL glass chamberat a concentration sufficient to yield oxygen consumption in the mediumof ≥10 pmol O₂ s⁻¹ mL⁻¹.

High-Resolution Respirometry—B (Used in Lactate Studies)

Real-time respirometric measurements were performed usinghigh-resolution oxygraphs (Oxygraph-2k, Oroboros Instruments, Innsbruck,Austria). The experimental conditions during the measurements were thefollowing: 37° C., 2 mL active chamber volume and 750 rpm stirrer speed.Chamber concentrations of O₂ were kept between 200-50 μM withreoxygenation of the chamber during the experiments as appropriate(Sjövall et al., 2013a). For data recording, DatLab software version 4and 5 were used (Oroboros Instruments, Innsbruck, Austria). Settings,daily calibration and instrumental background corrections were conductedaccording to the manufacturer's instructions. Respiratory measurementswere performed in either a buffer containing 0.5 mM EGTA, 3 mM MgCl₂, 60mM K-lactobionate, 20 mM Taurine, 10 mM KH₂PO₄, 20 mM HEPES, 110 mMsucrose and 1 g/L bovine serum albumin (MiR05) or phosphate bufferedsaline (PBS) with glucose (5 mM) and EGTA (5 mM), as indicated in thecorresponding sections. Respiratory values were corrected for the oxygensolubility factor both media (0.92) (Pesta and Gnaiger, 2012). Lactateproduction of intact human platelets was determined in PBS containing 10mM glucose. All measurements were performed at a platelet concentrationof 200×10⁶ cells per mL or a PBMC concentration of 5×10⁶ cells per mL.

Evaluation of Compounds

Four Typical Evaluation Protocols in Intact Cells are Utilized.

(1) Assay for Enhancement of Mitochondrial Energy Producing Function inCells with Inhibited Respiratory Complex I

Cells are placed in a buffer containing 110 mM sucrose, HEPES 20 mM,taurine 20 mM, K-lactobionate 60 mM, MgCl₂ 3 mM, KH₂PO₄ 10 mM, EGTA 0.5mM, BSA 1 g/l, pH 7.1. After baseline respiration with endogenoussubstrates is established, complex I is inhibited with Rotenone 2 μM.Compounds dissolved in DMSO are titrated in a range of 10 μM to 10 mMfinal concentration. Subsequently, cell membranes are permeabilised withdigitonin (1 mg/1*10⁶ plt) to allow entry of extracellularly releasedenergy substrate or cell impermeable energy substrates. After stabilizedrespiration, Succinate 10 mM is added as a reference to enablerespiration downstream of complex I. After the respiration stabilizedthe experiment is terminated by addition of Antimycin at finalconcentration 1 μg/mL and any residual non-mitochondrial oxygenconsumption is measured. An increase in respiration rate in thedescribed protocol is tightly coupled to ATP synthesis by oxidativephosphorylation unless cells are uncoupled (i.e. proton leak withoutproduction of ATP). Uncoupling is tested for by addition of the ATPsynthase inhibitor oligomycin (1-2 μg mL⁻¹) in a protocol 3 where theextent of uncoupling corresponds to the respiratory rate followingoligomycin addition.

(2) Assay for Enhancement and Inhibition of Mitochondrial EnergyProducing Function in Intact Cells

In the second protocol the same buffer is used as described above. Afterbasal respiration is established, the mitochondrial uncoupler FCCP isadded at a concentration of 2 nM to increase metabolic demand. Compoundsdissolved in DMSO are titrated in several steps from 10 μM to 10 mMfinal concentration in order to evaluate concentration range ofenhancement and/or inhibition of respiration. The experiment isterminated by addition of 2 μM Rotenone to inhibit complex I, revealingremaining substrate utilization downstream of this respiratory complex,and 1 μg/mL of the complex III inhibitor Antimycin to measurenon-mitochondrial oxygen consumption.

(3) Assay to Assess Uncoupling in Intact Cells

In the third protocol, the same buffer as described above is used. Afterbasal respiration is established, 1 mM of compound dissolved in DMSO isadded. Subsequently, the ATP-synthase-inhibitor Oligomycin is added. Areduction in respiration is a measure of how much of the oxygenconsumption that is coupled to ATP synthesis. No, or only a slight,reduction indicate that the compound is inducing a proton leak over theinner mitochondrial membrane. The uncoupler FCCP is then titrated toinduce maximum uncoupled respiration. Rotenone (2 μM) is then added toinhibit complex I, revealing remaining substrate utilization downstreamof this respiratory complex. The experiment is terminated by theaddition of 1 μg/mL of the complex III inhibitor Antimycin to measurenon-mitochondrial oxygen consumption.

(4) Assay for Enhancement of Mitochondrial Energy Producing Function inCells with Inhibited Respiratory Complex I in Human Plasma

Intact human blood cells are incubated in plasma from the same donor.After baseline respiration with endogenous substrates is established,complex I is inhibited with Rotenone 2 μM. Compounds dissolved in DMSOare titrated in a range of 10 μM to 10 mM final concentration. Theexperiment is terminated by addition of Antimycin at final concentration1 μg/mL and any residual non-mitochondrial oxygen consumption ismeasured.

Properties of Desired Compound in Respiration Assays

The ideal compound stimulates respiration in the described protocols inintact cells at low concentration without inhibitory effect on eithersuccinate stimulated respiration after permeabilization in protocol 1 orthe endogenous respiration in protocol 2. The concentration span betweenmaximal stimulatory effect and inhibition should be as wide as possible.After inhibition of respiration with mitochondrial toxins at ordownstream of complex III, respiration should be halted. Please refer toFIG. 1 and the listing below.

Desired properties of compounds:

-   -   maximum value of a reached at low drug concentration.    -   a substantially more than a″    -   a approaches b′    -   c approaches c′    -   d approaches d′

Compounds impermeable to the cellular membrane are identified in theassay as:

-   -   a approaches a′

Non mitochondrial oxygen consumption induced by drug candidate isidentified when

-   -   d more than d′

II. Assay for Prevention of Lactate Accumulation in Cells Exposed to aMitochondrial Complex I Inhibitor

Intact human platelets, white blood cells, fibroblasts, or other celltypes containing live mitochondria are incubated in phosphate bufferedsaline containing 10 mM glucose for 8 h with either of the complex Iinhibiting drugs metformin (10 mM), phenformin (0.5 mM) or rotenone (2μM). The inhibition of mitochondrial ATP production through oxidativephosphorylation by these compounds increases lactate accumulation byglycolysis. Lactate levels are determined every 2 h (or more frequent egevery 30 min) using the Lactate Pro™ 2 blood lactate test meter (Arkray,Alere AB, Lidingö, Sweden) or similar types of measurements. Incubationis performed at 37° C. pH is measured at start, after 4 and after 8 h(or more frequently) of incubation using a Standard pH Meter, e.g.PHM210 (Radiometer, Copenhagen, Denmark). Drug candidates are added tothe assay from start or following 30-60 min at concentrations within therange 10 μM-5 mM. The prevention of lactate accumulation is compared toparallel experiments with compound vehicle only, typically DMSO. Inorder to evaluate the specificity of the drug candidate, it is alsotested in combination with a down-stream inhibitor of respiration suchas the complex III inhibitor Antimycin at 1 μg/mL, which should abolishthe effect of the drug candidate and restore the production of lactate.The use of antimycin is therefore also a control for undue effects ofdrug candidates on the lactate producing ability of the cells used inthe assay. (See e.g. FIGS. 9, 10 and 11).

Data Analysis

Statistical analysis was performed using Graph Pad PRISM software(GraphPad Software version 6.03, La Jolla, Calif., USA). Allrespiratory, lactate and pH data are expressed as mean±SEM. Ratios areplotted as individuals and means. One-way ANOVA was used for one-factorcomparison of three or more groups (concentration of drugs) and two-waymixed model ANOVA was used for two-factor comparison (time andconcentration of drugs/treatment) of three or more groups. Post-hoctests to compensate for multiple comparisons were done according toHolm-Sidak. Correlations were expressed as r² and P-values. Standardnon-linear curve fitting was applied to calculate half maximalinhibitory concentration (IC₅₀) values. Results were consideredstatistically significant for P<0.05.

Properties of Desired Compound in Cellular Lactate Accumulation Assay

-   -   (1) The ideal compound prevents the lactate accumulation induced        by complex I inhibition, i.e. the lactate accumulation        approaches a similar rate as that in non complex I-inhibited        cells. (2) The prevention of lactate accumulation is abolished        by a downstream respiratory inhibitor such as Antimycin.

III. Assay for Prevention of Lactate Accumulation and EnergeticInhibition in an Acute Metabolic Crisis Model in Pig

Lead drug candidates will be tested in a proof of concept in vivo modelof metabolic crisis due to mitochondrial dysfunction at complex I. Themodel mimics severe conditions that can arise in children with geneticmutations in mitochondrial complex I or patients treated and overdosedwith clinically used medications such as metformin, which inhibitscomplex I when accumulated in cells and tissues.

Female landrace pigs are used in the study. They are anaesthetized,taken to surgery in which catheters are placed for infusions andmonitoring activities. A metabolic crisis is induced by infusion of themitochondrial complex I inhibitor rotenone at a rate of 0.25 mg/kg/hduring 3 h followed by 0.5 mg/kg/h infused during one hour (vehicleconsisting of 25% NM P/4% polysorbate 80/71% water). Cardiovascularparameters such as arterial blood pressure is measured continuouslythrough a catheter placed in the femoral artery. Cardiac output (CO) ismeasured and recorded every 15 minutes by thermo-dilution, and pulmonaryartery pressure (PA, systolic and diastolic), central venous pressure(CVP), and SvO₂ is recorded every 15 min and pulmonary wedge pressure(PCWP) every 30 min from a Swan-Ganz catheter. Indirect calorimetry isperformed e.g. by means of a Quark RMR ICU option (Cosmed, Rome, Italy)equipment. Blood gases and electrolytes are determined in both arterialand venous blood collected from the femoral artery and Swan-Ganzcatheters and analysed with use of an ABL725 blood gas analyser(Radiometer Medical Aps, Brønshøj, Denmark). Analyses include pH, BE,Hemoglobin, HCO₃, pO₂, pCO₂, K⁺, Na⁺, Glucose and Lactate.

Properties of Desired Compound in a Proof of Concept In Vivo Model ofMetabolic Crisis

The ideal compound should reduce the lactate accumulation and pHdecrease in pigs with metabolic crisis induced by complex I inhibition.The energy expenditure decrease following complex I inhibition should beattenuated. The compound should not induce any overt negative effects asmeasured by blood and hemodynamic analyses.

Metabolomics Method

White blood cells or platelets are collected by standard methods andsuspended in a MiR05, a buffer containing 110 mM sucrose, HEPES 20 mM,taurine 20 mM, K-lactobionate 60 mM, MgCl₂ 3 mM, KH₂PO₄ 10 mM, EGTA 0.5mM, BSA 1 g/I, with or without 5 mM glucose, pH 7.1. The sample isincubated with stirring in a high-resolution oxygraph (Oxygraph-2k,Oroboros Instruments, Innsbruck, Austria) at a constant temperature of37° C.

After 10 minutes rotenone in DMSO is added (2 μM) and incubationcontinued. Following a further 5 minutes test compound in DMSO is added,optionally with further test compound after and a further period ofincubation. During the incubation O₂ consumption is measured inreal-time.

At the end of the incubation the cells are collected by centrifugationand washed in 5% mannitol solution and extracted into methanol. Anaqueous solution containing internal standard is added and the resultantsolution treated by centrifugation in a suitable microfuge tube with afilter.

The resulting filtrate is dried under vacuum before CE-MS analysis toquantify various primary metabolites by the method of Ooga et al (2011)and Ohashi et al (2008).

In particular the levels of metabolite in the TCA cycle and glycolysisare assessed for the impact of compounds of the invention.

Ooga et al, Metabolomic anatomy of an animal model revealing homeostaticimbalances in dyslipidaemia, Molecular Biosystems, 2011, 7, 1217-1223Ohashi et al, Molecular Biosystems, 2008, 4, 135-147

Materials

Unless otherwise indicated, all reagents used in the examples below areobtained from commercial sources.

EXAMPLES Example 1

Succinyl chloride (0.1 mol) and triethylamine (0.4 mol) is dissolved inDCM and cysteine is added. The reaction is stirred at room temperature.The reaction is added to aqueous dilute hydrochloric acid and then iswashed water and brine. The organic layers are dried over magnesiumsulfate and reduced in vacuo. The target compound is the purified bysilica gel chromatography.

Example 2—Synthesis of S,S-bis(2-propionamidoethyl) butanebis(thioate)(NV038, 01-038)

To a solution of cysteamine hydrochloride (5.0 g, 44 mmol) in CH₃OH (50mL) was added Et₃N (4.4 g, 44 mmol), followed by (Boc)₂O (10.5 g, 48.4mmol) and the mixture was stirred at room temperature for 1h. Thereaction mixture was concentrated in vacuo. The obtained residue wasdissolved in CH₂Cl₂, washed with 2M HCl aqueous solution and brine,dried over Na₂SO₄, filtered and evaporated to yield tert-butyl2-mercaptoethylcarbamate as a colorless oil which was used in the nextstep without further purification.

tert-Butyl 2-mercaptoethylcarbamate (9.8 g, 55.0 mmol) and Et₃N (5.6 g,55.0 mmol) were dissolved in CH₂Cl₂ (100 mL), the mixture cooled to 0°C., succinyl chloride (2.1 g, 13.8 mmol) was added with dropwise. Thenthe mixture was stirred at room temperature for 2h. The reaction mixtureconcentrated and the residue was purified by column chromatography(petrol ether/EtOAc=1/10 to 1/1).S,S-bis(2-(tert-butoxycarbonylamino)ethyl) butanebis(thioate) wasobtained as a white solid.

A mixture of S,S-bis(2-(tert-butoxycarbonylamino)ethyl)butanebis(thioate) (2.0 g, 4.58 mmol) and TFA (10 mL) in CH₂Cl₂ (10 mL)was stirred at room temperature for 4 hours. The reaction mixture wasconcentrated to yield S,S-bis(2-aminoethyl) butanebis(thioate) as ayellow oil which was used in the next step without further purification.

S,S-bis(2-aminoethyl) butanebis(thioate) (1.1 g, 4.58 mmol) and Et₃N(1.4 g, 13.74 mmol) were dissolved in CH₂Cl₂ (15 mL), the mixture cooledto 0° C., propionyl chloride (0.9 g, 10.07 mmol) was added withdropwise. Then the mixture was stirred at room temperature for 3 hours.The reaction mixture concentrated and the residue was purified bypreparative TLC (CH₂Cl₂/MeOH=15/1). S,S-bis(2-Propionamidoethyl)butanebis(thioate) was obtained as a white solid.

Example 3—synthesis of(R)-4-(2-carboxy-2-propionamidoethylthio)-4-oxobutanoic acid (NV-041,01-041)

To a mixture of L-cysteine (2.00 g, 16.5 mmol) in THF/H₂O (8 mL/2 mL)was added NaOAc (2.70 g, 33.0 mmol). The mixture was stirred at roomtemperature for 20 min. The reaction was cooled to 5° C. beforepropionic anhydride (2.30 g, 17.6 mmol) was added dropwise. The reactionmixture was stirred at room temperature overnight and then heated toreflux for 4 hours. The reaction mixture was cooled and acidified to pH5 by adding 4N HCl. The resulting solution was evaporated under reducedpressure to remove THF. The residue was purified by prep-HPLC (elutingwith H₂O (0.05% TFA) and CH₃CN) to give 1.00 g of(R)-3-mercapto-2-propionamidopropanoic acid as colourless oil.

A solution of (R)-3-mercapto-2-propionamidopropanoic acid (1.00 g, 5.65mmol), succinic anhydride (565 mg, 5.65 mmol) and Et₃N (572 mg, 5.65mmol) in 10 mL of THF was heated under reflux overnight. The reactionmixture concentrated and the residue was purified by preparative-HPLC(eluting with H₂O (0.05% TFA) and CH₃CN) to yield(R)-4-(2-carboxy-2-propionamidoethylthio)-4-oxobutanoic acid as acolourless oil.

Example 4

Step 1

Triethylamine (0.24 mol) is added to a solution of N-acetylcysteamine(0.2 mol) in DCM. 4-Chloro-4-oxobutanoic acid (0.1 mol) is addeddropwise, and the reaction mixture is stirred at room temperature. Themixture is added to aqueous dilute hydrochloric acid and is extractedwith ethyl acetate, and then is washed water and brine. The organiclayers are dried over magnesium sulfate and reduced in vacuo.

Step 2

The product of step 3 (0.1 mol), acetic acid 1-bromoethyl ester (0.1mol) and caesium carbonate (0.12 mol) is suspended in DMF and stirred at60° C. under an inert atmosphere. The suspension is allowed to cool toroom temperature and ethyl acetate added and is washed successively withaqueous dilute hydrochloric acid and water. The organics are dried overmagnesium sulfate and reduced in vacuo. The residue is purified bycolumn chromatography.

Example 5

Step 1

Triethylamine (0.24 mol) is added to a solution of N-acetylcysteamine(0.2 mol) in DCM. 4-Chloro-4-oxobutanoic acid (0.1 mol) is addeddropwise, and the reaction mixture is stirred at room temperature. Themixture is added to aqueous dilute hydrochloric acid and is extractedwith ethyl acetate, and then is washed water and brine. The organiclayers are dried over magnesium sulfate and reduced in vacuo.

Step 2

Dimethylamine (0.1 mol) and triethylamine (0.1 mol) are diluted indichloromethane, the solution is cooled to 0° C. and 2-chloropropionylchloride (0.1 mol) in DCM is added and the solution is allowed to warmto room temperature and is left to stir under an inert atmosphere. Thesolution is washed with water. The organics are combined and thevolatiles are removed in vacuo. The residue is purified by silica gelchromatography.

Step 3

2-Chloro-N,N-dimethyl-propionamide (0.1 mol), the product of step 1 (0.1mol), caesium carbonate (0.1 mol), and sodium iodide (0.01 mol) issuspended in DMF and the suspension stirred at 80° C. under an inertatmosphere. The suspension is cooled to room temperature, is dilutedwith ethyl acetate and is washed with water. The organics are reduced invacuo. The residue is purified by silica gel chromatography to yield thetarget compound.

Example 6—synthesis of 4-oxo-4-(2-propionamidoethylthio)butanoic acid(NV114, 01-114)

Propionic anhydride (11.7 g, 89.7 mmol) and aqueous KOH (8 M, tomaintain pH=8) were added dropwise to a stirred solution of cysteaminehydrochloride (3.40 g, 30.0 mmol) in 24 mL of water. The mixture wasneutralized by adding 2N HCl and stirred for 1 hour at room temperature.The solution was cooled with an ice bath and solid KOH (6.00 g, 105mmol) was added slowly. The mixture was stirred for 50 minutes at roomtemperature. After saturated with NaCl and neutralized with 6N HCl, themixture was extracted with CH₂Cl₂ (4×30 mL). The combined CH₂Cl₂extracts were dried (Na₂SO₄) and concentrated in vacuo to giveN-(2-mercaptoethyl)propionamide as colourless oil, which was used fornext step without further purification.

A solution of N-(2-mercaptoethyl)propionamide (2.00 g, 15.0 mmol),succinic anhydride (1.50 g, 15.0 mmol) and Et₃N (1.50 g, 15.0 mmol) in20 mL of THF was heated under reflux overnight. The reaction mixture wasconcentrated and the residue was purified by preparative-HPLC (elutingwith H₂O (0.05% TFA) and CH₃CN) to yield4-oxo-4-(2-propionamidoethylthio)butanoic acid as colourless oil.

Example 7—synthesis of 4-(2-acetamidoethylthio)-4-oxobutanoic acid(NV108, 01-108)

Acetic anhydride (8.48 mL, 90.0 mmol) and aqueous KOH (8 M, to maintainpH=8) were added dropwise to a stirred solution of cysteaminehydrochloride (3.40 g, 30.0 mmol) in 24 mL of water. The pH was thenadjusted to 7 with adding 2N HCl. The mixture was stirred for 1 hour atroom temperature, and then the solution was cooled with an ice bath. Tothe above solution, solid KOH (6.0 g, 105 mmol) was added slowly, andthe resulting mixture was stirred for 50 minutes at room temperature.After saturated with NaCl and neutralized with 6N HCl, the mixture wasextracted with CH₂Cl₂ (4×30 mL). The combined CH₂Cl₂ extracts were dried(Na₂SO₄) and concentrated in vacuo to give N-(2-mercaptoethyl)acetamideas colourless oil, which was used for next step without furtherpurification.

A solution of N-(2-mercaptoethyl)acetamide (1.50 g, 12.7 mmol), succinicanhydride (1.3 g, 12.7 mmol) and Et₃N (1.3 g, 12.7 mmol) in 20 mL of THFwas heated under reflux overnight. The reaction mixture was concentratedand the residue was purified by preparative HPLC (eluting with H₂O(0.05% TFA) and CH₃CN) to yield 4-(2-acetamidoethylthio)-4-oxobutanoicacid as colourless oil.

Example 8—The synthesis of(R)-3-(4-((R)-2-carboxy-2-propionamidoethylthio)-4-oxobutanoylthio)-2-propionamidopropanoicacid (NV099, 01-099)

To a mixture of N-hydroxysuccinimide (3.00 g, 26.1 mmol) and Et₃N (3.20g, 31.3 mmol) in CH₂Cl₂ (60 mL) was added dropwise succinyl chloride(2.00 g, 13.0 mmol). The mixture was stirred at room temperature for 3hours before diluted with water (60 mL). The resulting suspension wasfiltered, washed with water and CH₂Cl₂. The cake was collected and driedto give bis(2,5-dioxopyrrolidin-1-yl) succinate as a grey solid.

A mixture of N-(2-mercaptoethyl)propionamide (400 mg, 2.26 mmol),bis(2,5-dioxopyrrolidin-1-yl) succinate (353 mg, 1.13 mmol) and TEA (286mg, 2.83 mmol) in 3.0 mL of CH₃CN was stirred at room temperature for 2hours. The clear reaction solution was purified by preparative-HPLC(eluting with H₂O (0.05% TFA) and CH₃CN) directly to yield(R)-3-(4-((R)-2-carboxy-2-propionamidoethylthio)-4-oxobutanoylthio)-2-propionamidopropanoicacid as colorless oil.

Example 9—Synthesis of(R)-4-(1-carboxy-2-(propionylthio)ethylamino)-4-oxobutanoic acid (NV122,01-122)

To a mixture of (R)-3-mercapto-2-propionamidopropanoic acid (1.00 g,8.25 mmol) and propionic acid (1.0 mL) in CHCl₃ (10 mL) were addedpropionic anhydride (1.13 g, 8.67 mmol) dropwise. The reaction mixturewas heated to reflux overnight. The reaction mixture was cooled andsuccinic anhydride (1.00 g, 9.99 mmol) was added. The mixture wasrefluxed overnight before concentrated under reduced pressure. Theresidue was purified by prep-HPLC (eluting with H₂O (0.05% TFA) andCH₃CN) to yield(R)-4-(1-carboxy-2-(propionylthio)ethylamino)-4-oxobutanoic acid as anoff-white solid.

Example 10—The synthesis of4-(1-acetamido-2-methylpropan-2-ylthio)-4-oxobutanoic acid (NV188,01-188)

To a stirred solution of cysteamine hydrochloride (2.00 g, 14.1 mmol) in15 mL of water was added acetic anhydride (4.30 g, 42.4 mmol) andaqueous KOH (8 M, to maintain pH=8) dropwise. The mixture was thenneutralized by adding 2N HCl and stirred for 1 hour at room temperature.To the solution cooled with an ice bath was added slowly solid KOH (2.80g, 49.4 mmol) and the mixture was stirred for 50 minutes at roomtemperature. After saturated with NaCl and neutralized with 6N HCl, themixture was extracted with CH₂Cl₂ twice. The combined CH₂Cl₂ extractswere dried (Na₂SO₄) and concentrated in vacuo to yieldN-(2-mercapto-2-methylpropyl)acetamide as a white solid which was usedfor next step without further purification.

A solution of N-(2-mercapto-2-methylpropyl)acetamide (400 mg, 2.72mmol), succinic anhydride (326 mg, 3.26 mmol) and Et₃N (330 mg, 3.26mmol) in 6 mL of THF was heated under overnight. The reaction mixturewas concentrated and the residue was purified by preparative-HPLC(eluting with H₂O (0.05% TFA) and CH₃CN) to yield4-(1-acetamido-2-methylpropan-2-ylthio)-4-oxobutanoic acid as yellowoil.

Example 11—The synthesis ofS,S-bis((R)-3-(diethylamino)-3-oxo-2-propionamidopropyl)butanebis(thioate) (NV185, 01-185)

To a solution of (R)-3-mercapto-2-propionamidopropanoic acid (5.00 g,28.0 mmol) in DMF (50 mL) was added triphenylmethyl chloride (8.70 g,31.0 mmol) at 0° C. The mixture was stirred at 0° C. for 30 min and thenwarmed to room temperature overnight. The mixture was treated with waterand extracted with EtOAc twice. The combined organic layers were washedwith brine, dried over Na₂SO₄ and concentrated under reduced pressure.The residue was purified by silica gel column chromatography(CH₂Cl₂/MeOH=80/1˜50/1) to yield(R)-2-propionamido-3-(tritylthio)propanoic acid as a white solid.

To a stirred solution of (R)-2-propionamido-3-(tritylthio)propanoic acid(1.7 g, 4.0 mmol) in CH₂Cl₂ (50 mL) was added DCC (1.7 g, 8.0 mmol) andHOBT (0.50 g. 4.0 mmol) at room temperature. The mixture was stirred atroom temperature for 1 h and then diethylamine (0.80 g, 8.0 mmol) wasadded. The mixture was stirred at room temperature overnight. Themixture was washed with water, dried over Na₂SO₄ and concentrated underreduced pressure to give the crude product which was purified by silicagel column chromatography (EtOAc/petrol ether=1/6˜1/1) to yield(R)-N,N-diethyl-3-mercapto-2-propionamidopropanamide as yellow oil.

To a solution of (R)-N,N-diethyl-3-mercapto-2-propionamidopropanamide(400 mg, 0.800 mmol) in CH₂Cl₂ (10 mL) at 0° C. was added TFA (1 mL) andi-Pr3SiH (253 mg, 1.60 mmol). The mixture was warmed to room temperatureand stirred for 2 hours. The solution was evaporated under reducedpressure. The residue was purified by preparative-HPLC (eluting with H₂O(0.5% TFA) and CH₃CN) to yield(R)-N,N-diethyl-3-mercapto-2-propionamidopropanamide as yellow oil.

A mixture of (R)-N,N-diethyl-3-mercapto-2-propionamidopropanamide (150mg, 0.600 mmol), Et₃N (242 mg, 2.40 mmol) andbis(2,5-dioxopyrrolidin-1-yl) succinate (94 mg, 0.30 mmol) in CH₃CN (100mL) was stirred at room temperature overnight. The mixture wasevaporated under reduced pressure. The residue was purified bypreparative HPLC (eluting with H₂O (0.5% TFA) and CH₃CN) to yieldS,S-bis((R)-3-(diethylamino)3-oxo-2-propionamidopropyl)butanebis(thioate) (36% yield) as a yellow solid.

Example 12—The synthesis of4-(2-(2-(diethylamino)-2-oxoethoxy)ethylthio)-4-oxobutanoic acid (NV193,01-193)

To a solution of 2-bromoacetyl bromide (4.00 g, 20.0 mmol) and DIPEA(2.60 g, 20 mmol) in CH₂Cl₂ (50 mL) was added dropwise diethylamine(1.60 g, 20.0 mmol) at 0° C. The mixture was stirred at 0° C. for 30min. The solution was evaporated under reduced pressure to removeCH₂Cl₂. The residue was purified by silica gel column chromatography(EtOAc/petrol ether=1/5˜1/2) to yield 2-bromo-N,N-diethylacetamide asyellow oil.

A solution of 2-mercaptoethanol (2.50 g, 32.0 mmol), triphenylmethylchloride (10.7 g, 38.4 mmol) in 100 mL of THF was heated under refluxovernight. The reaction mixture was concentrated and the residue waspurified by silica gel column chromatography (EtOAc/petrolether=1/5˜1/1) to yield 2-(2,2,2-triphenylethylthio)ethanol as a whitesolid.

To a solution of 2-(2,2,2-triphenylethylthio)ethanol (3.50 g, 10.9 mmol)in THF (30 mL) was added NaH (0.500 g, 13.0 mmol, 60% in oil) inportions at 0° C. The reaction mixture was stirred at 0° C. for 1 hour.Then a solution of 2-bromo-N,N-diethylacetamide (2.1 g, 10.9 mmol) inTHF (5 mL) was added dropwise. The resulting mixture was warmed to roomtemperature over 2 hours. The mixture was quenched with water andextracted with EtOAc twice. The combined organic layers were washed withbrine, dried over Na₂SO₄ and concentrated under reduced pressure. Theresidue was purified by silica gel column chromatography (EtOAc/petrolether=1/5˜1/2) to yield N,N-diethyl-2-(2-(tritylthio)ethoxy)acetamide asa white solid.

To a solution of N,N-diethyl-2-(2-(tritylthio)ethoxy)acetamide (2.70 g,6.30 mmol) in CH₂Cl₂ (20 mL) was added TFA (2 mL) and i-Pr₃SiH (2.00 g,12.6 mmol) at 0° C. The mixture was warmed to room temperature andstirred for 2 hours. The solution was evaporated under reduced pressureto remove CH₂Cl₂. The residue was purified by silica gel columnchromatography (EtOAc/petrol ether=1/5˜1/1) to yieldN,N-diethyl-2-(2-mercaptoethoxy)acetamide as colorless oil.

A solution of N,N-diethyl-2-(2-mercaptoethoxy)acetamide (356 mg, 1.90mmol), succinic anhydride (200 mg, 2.10 mmol) and Et₃N (300 mg, 2.90mmol) in 10 mL of THF was stirred at reflux overnight. The reactionmixture was concentrated in vacuo and the residue was purified bypreparative HPLC (eluting with H₂O (0.5% TFA) and CH₃CN) to yield4-(2-(2-(diethylamino)-2-oxoethoxy)ethylthio)-4-oxobutanoic acid ascolorless oil.

Example 13—The synthesis of (R)-methyl3-(4-((R)-3-methoxy-3-oxo-2-propionamidopropylthio)-4-oxobutanoylthio)-2-propionamidopropanoate(NV205, 01-205)

A mixture of(R)-3-(4-((R)-2-carboxy-2-propionamidoethylthio)-4-oxobutanoylthio)-2-propionamidopropanoicacid (300 mg, 0.69 mmol), CH₃I (293 mg, 2.06 mmol) and K₂CO₃ (475 mg,3.44 mmol) in 4.0 mL of DMF was stirred at room temperature overnight.The reaction mixture was filtered and the filtrate was purified bypreparative-HPLC (eluting with H₂O (0.05% TFA) and CH₃CN) directly toyield (R)-methyl3-(4-((R)3-methoxy-3-oxo-2-propionamidopropylthio)-4-oxobutanoylthio)-2-propionamidopropanoateas an off-white solid.

Example 14—Synthesis of NV189

A mixture of N-(2-mercapto-2-methylpropyl)acetamide (400 mg, 2.72 mmol),bis(2,5-dioxopyrrolidin-1-yl) succinate (339 mg, 1.09 mmol) and Et₃N(550 mg, 5.44 mmol) in 6 mL of CH₃CN was stirred at room temperatureovernight. The reaction mixture was concentrated and the residue waspurified by preparative HPLC (eluting with H₂O (0.05% TFA) and CH₃CN) toyield NV189 as an off-white solid.

Example 15—Synthesis of S,S-bis(2-(2-(diethylamino)-2-oxoethoxy)ethyl)butane-bis(thioate) (NV195, 01-195)

To a solution of N,N-diethyl-2-(2-mercaptoethoxy)acetamide (438 mg, 2.3mmol) in CH₃CN (10 mL) was added bis(2,5-dioxopyrrolidin-1-yl) succinate(374 mg, 1.2 mmol) and Et₃N (232 mg, 2.3 mmol). The mixture was stirredat room temperature overnight. The reaction mixture was concentrated invacuo and the residue was purified by preparative HPLC (eluting with H₂O(0.5% TFA) and CH₃CN) to yieldS,S-bis(2-(2-(diethylamino)-2-oxoethoxy)ethyl) butanebis(thioate) as acolorless oil.

Example 16—Synthesis of NV206

A mixture of(R)-3-(4-((R)-2-carboxy-2-propionamidoethylthio)-4-oxobutanoylthio)-2-propionamidopropanoicacid (400 mg, 0.916 mmol), CH₃I (156 mg, 1.1 mmol) and K₂CO₃ (190 mg,1.37 mmol) in 4 mL of DMF was stirred at room temperature for 6 hours.The reaction mixture was filtered and the filtrate was purified byprep-HPLC (eluting with H₂O (0.05% TFA) and CH₃CN) directly to yieldNV206 as a colorless gum.

Example 17

Results of Biological Experiments

The compounds given in the following table were subject to the assays(1)-(4) mentioned under the heading I. Assay for evaluating enhancementand inhibition of mitochondrial energy producing function in intactcells. In the following table the results are shown, which indicate thatall compounds tested have suitable properties. Importantly, allcompounds show specific effect on CII-linked respiration as seen fromscreening protocols 1 and 4, as well as a convergent effect, withCl-substrates available, as seen in assay 2.

Results from Screening Protocols 1-4

Compound numbers as set out in Examples 1-16.

Convergent Convergent CII Comnpound NV (Routine) (FCCP) (plasma) CIIUncoupling Toxicity 01-193 (++) + (+) + + 5 mM 01-188 +++ +++ + + (+) 5mM 01-185 (+) + + + (+) 2 mM 01-205 +++ ++ + ++ (+) 5 mM 01-114 +++ ++ +++ (+) 10 mM 01-041 + +++ + ++ (+) 5 mM 01-108 ++ ++ (+) (++) + 10 mM

Legend: Convergent (Routine)—the increase in mitochondrial oxygenconsumption induced by the compound under conditions described inscreening assay 3; Convergent (FCCP)—the increase in mitochondrialoxygen consumption induced by the compound under conditions described inscreening assay 2 (uncoupled conditions); Convergent (plasma)—theincrease in mitochondrial oxygen consumption induced by the compound incells with inhibited complex I incubated in human plasma, as describedin screening assay 4; CII—the increase in mitochondrial oxygenconsumption induced by the compound in cells with inhibited complex I asdescribed in screening assay 1; Uncoupling—the level of oxygenconsumption after addition of oligomycin as described in screening assay3. The response in each parameter is graded either +, ++ or +++ inincreasing order of potency. Brackets [( )] indicate an intermediateeffect, i.e. (+++) is between ++ and +++. Toxicity—the lowestconcentration during compound titration at which a decrease in oxygenconsumption is seen as described in screening assay 2.

Examples 18-20

Metformin Studies

In the metformin study the following compounds were used (and which arereferred to in the figures). The compounds are described in WO2014/053857.

Sample Acquisition and Preparation

The study was performed with approval of the regional ethical reviewboard of Lund University, Sweden (ethical review board permit no.2013/181). Venous blood from 18 healthy adults (11 males and 7 females)was drawn in K₂EDTA tubes (BD Vacutainer® Brand Tube with dipotassiumEDTA, BD, Plymouth, UK) according to clinical standard procedure afterwritten informed consent was acquired. For platelet isolation the wholeblood was centrifuged (Multifuge 1 S-R Heraeus, Thermo FisherScientifics, Waltham, USA) at 500 g at room temperature (RT) for 10 min.Platelet-rich plasma was collected to 15 mL falcon tubes and centrifugedat 4600 g at RT for 8 min. The resulting pellet was resuspended in 1-2mL of the donor's own plasma. PBMCs were isolated using Ficol gradientcentrifugation (Boyum, 1968). The blood remaining after isolation ofplatelets was washed with an equal volume of physiological saline andlayered over 3 mL of Lymphoprep™. After centrifugation at 800 g at RT(room temperature) for 30 min the PBMC layer was collected and washedwith physiological saline. Following a centrifugation at 250 g at RT for10 min the pellet of PBMCs was resuspended in two parts of physiologicalsaline and one part of the donor's own plasma. Cell count for both PBMCsand platelets were performed using an automated hemocytometer (SwelabAlfa, Boule Medical AB, Stockholm, Sweden).

Aim of Study Reported in Examples 18-19

Metformin induces lactate production in peripheral blood mononuclearcells and Platelets Through Specific Mitochondrial Complex I Inhibition

Metformin is a widely used anti-diabetic drug associated with the rareside-effect of lactic acidosis, which has been proposed to be linked todrug-induced mitochondrial dysfunction. Using respirometry, the aim ofthe study reported in Examples 1-2 below was to evaluate mitochondrialtoxicity of metformin to human blood cells in relation to that ofphenformin, a biguanide analog withdrawn in most countries due to a highincidence of lactic acidosis.

Aim of the Study Reported in Example 20

The aim is to investigate the ability of succinate prodrugs to alleviateor circumvent undesired effects of metformin and phenformin.

Example 18A

Effects of Metformin and Phenformin on Mitochondrial Respiration inPermeabilized Human Platelets

In order to investigate the specific target of biguanide toxicity, aprotocol was applied using digitonin permeabilization of the blood cellsand sequential additions of respiratory complex-specific substrates andinhibitors in MiR05 medium. After stabilization of routine respiration,i.e. respiration of the cells with their endogenous substrate supply andATP demand, metformin, phenformin or their vehicle (double-deionizedwater) were added. A wide concentration range of the drugs was applied;0.1, 0.5, 1, and 10 mM metformin and 25, 100 and 500 μM phenformin.After incubation with the drugs for 10 min at 37° C., the platelets werepermeabilized with digitonin at a previously determined optimaldigitonin concentration (1 μg 10⁻⁶ platelets) to induce maximal cellmembrane permeabilization without disruption of the mitochondrialfunction and allowing measurements of maximal respiratory capacities(Sjövall et al. (2013a). For evaluation of complex I-dependent oxidativephosphorylation capacity (OXPHOS_(CI)) first, the NADH-linked substratespyruvate and malate (5 mM), then ADP (1 mM) and, at last, the additionalcomplex I substrate glutamate (5 mM) were added sequentially.Subsequently the FADH₂-linked substrate succinate (10 mM) was given todetermine convergent complex I- and II-dependent OXPHOS capacity(OXPHOS_(CI+II)). LEAKI+II state, a respiratory state where oxygenconsumption is compensating for the back-flux of protons across themitochondrial membrane (Gnaiger, 2008), was assessed by addition of theATP-synthase inhibitor oligomycin (1 μg mL⁻¹). Maximal uncoupledrespiratory electron transport system capacity supported by convergentinput through complex I and II (ETS_(CI+II)) was evaluated by subsequenttitration with the protonophore carbonyl-cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP). Addition of the complex I inhibitor rotenone (2μM) revealed complex II-dependent maximal uncoupled respiration(ETS_(CII)). The complex III inhibitor antimycin (1 μg mL⁻¹) was thengiven to reveal residual oxygen consumption (ROX). Finally, theartificial complex IV substrate N,N,N′,N′-tetramethyl-p-phenylenediaminedihydrochloride (TMPD, 0.5 mM) was added and the complex IV inhibitorsodium azide (10 mM) was given to measure complex IV activity andchemical background, respectively. Complex IV activity was calculated bysubtracting the sodium azide value from the TMPD value. With exceptionof complex IV activity, all respiratory states were measured atsteady-state and corrected for ROX. Complex IV activity was measuredafter ROX determination and not at steady-state. The integrity of theouter mitochondrial membrane was examined by adding cytochrome c (8 μM)during OXPHOS_(CI+II) in presence of vehicle, 100 mM metformin or 500 μMphenformin.

Example 18B

Effect of Metformin on Mitochondrial Respiration in Permeabilized HumanPeripheral Blood Mononuclear Cells and on Mitochondrial Respiration inIntact Human Platelets

For analysis of respiration of permeabilized PBMCs in response tometformin (0.1, 1 and 10 mM) the same protocol as for permeabilizedplatelets was used, except the digitonin concentration was adjusted to 6μg 10⁻⁶ PBMCs (Sjövall et al., 2013b).

Results

Respiration using complex I substrates was dose-dependently inhibited bymetformin in both permeabilized human PBMCs and platelets (FIG. 1).OXPHOS_(CI) capacity decreased with increasing concentrations ofmetformin compared to controls with near complete inhibition at 10 mM(−81.47%, P<0.001 in PBMCs and −92.04%, P<0.001 in platelets), resultingin an IC₅₀ of 0.45 mM for PBMCs and 1.2 mM for platelets. Respiratorycapacities using both complex I- and complex II-linked substrates,OXPHOS_(CI+II) and ETS_(CI+II), were decreased similarly to OXPHOS_(CI)by metformin as illustrated by the representative traces ofsimultaneously measured O₂ consumption of vehicle-treated and 1 mMmetformin-treated permeabilized PBMCs (FIG. 5a ). In contrast, ETS_(CII)capacity and complex IV activity did not change significantly inpresence of metformin compared to controls in either cell type (FIG. 5b,c ) and neither did LEAK_(I+II) respiration (the respiratory state whereoxygen consumption is compensating for the back-flux of protons acrossthe mitochondrial membrane, traditionally denoted state 4 in isolatedmitochondria, data not shown). The mitochondrial inhibition of complex Iinduced by metformin did not seem to be reversible upon extra- andintracellular removal of the drug by washing and permeabilizing thecells, respectively. Although the severity of the insult of complex Iinhibition was attenuated by removal (probably attributed to a shorterexposure time of the drug) platelets did not regain routine and maximalmitochondrial function comparable to control (data not shown).Phenformin likewise inhibited OXPHOS_(CI) (FIG. 6), OXPHOS_(CI+II) andETS_(CI+II) but not ETS_(CII) or respiration specific to complex IV(data not shown). Phenformin demonstrated a 20-fold more potentinhibition of OXPHOS_(CI) in permeabilized platelets than metformin(IC₅₀ 0.058 mM and 1.2 mM, respectively) (FIG. 2). Metformin andphenformin did not induce increased respiration following administrationof cytochrome c and hence did not disrupt the integrity of the outermitochondrial membrane.

After stabilization of routine respiration in MiR05 medium, eithervehicle (double-deionized water) or 1, 10 and 100 mM metformin wasadded. Routine respiration was followed for 60 min at 37° C. before theATP-synthase inhibitor oligomycin (1 μg mL⁻¹) was added to assess LEAKrespiration. Maximal uncoupled respiratory electron transport systemcapacity supported by endogenous substrates (ETS) was reached bytitration of FCCP. Respiration was sequentially blocked by the complex Iinhibitor rotenone (2 μM), the complex III inhibitor antimycin (1 μgmL⁻¹) and the complex IV inhibitor sodium azide (10 mM) to assess ROX,which all respiration values were corrected for. In an additionalexperiment, whole blood was incubated in K₂EDTA tubes with differentmetformin concentrations (0.1, 0.5 and 1 mM) over a period of 18 h priorto isolation of platelets and analyses of respiration.

Results

In intact human platelets, metformin decreased routine respiration in adose- and time-dependent manner (FIG. 7a ). When exposed to eithermetformin or vehicle the platelets showed a continuous decrease inroutine respiration over time. After 60 min the routine respiration wasreduced by −14.1% in control (P<0.05), by −17.27% at 1 mM (P<0.01), by−28.61% at 10 mM (P<0.001), and by −81.78% at 100 mM of metformin(P<0.001) compared to the first measurement after addition. Metformin at100 mM decreased routine respiration significantly compared to controlalready after 15 min of exposure (−39.77%, P<0.01). The maximaluncoupled respiration of platelets (the protonophore-titrated ETScapacity) after 60 min incubation, was significantly inhibited by 10 mM(−23.86%, P<0.05) and 100 mM (−56.86%, P<0.001) metformin (FIG. 3b ).LEAK respiration in intact cells was not significantly changed bymetformin incubation (data not shown). When whole blood was incubated ata metformin concentrations of 1 mM over 18 h routine respiration ofintact human platelets was reduced by 30.49% (P<0.05).

Example 19

Effect of Metformin and Phenformin on Lactate Production and pH ofIntact Human Platelets

Platelets were incubated for 8 h with either metformin (1 mM, 10 mM),phenformin (0.5 mM), rotenone (2 μM), or the vehicle for rotenone(DMSO). Lactate levels were determined every 2 h (n=5) using the LactatePro™ 2 blood lactate test meter (Arkray, Alere AB, Lidingö, Sweden)(Tanner et al., 2010). Incubation was performed at 37° C. at a stirrerspeed of 750 rpm, and pH was measured at start, after 4 and after 8 h ofincubation (n=4) using a PHM210 Standard pH Meter (Radiometer,Copenhagen, Denmark).

Results

Lactate production increased in a time- and dose-dependent manner inresponse to incubation with metformin and phenformin in human platelets(FIG. 8). Compared to control, metformin—(1 and 10 mM), phenformin—(0.5mM), and rotenone—(2 μM) treated platelets all produced significantlymore lactate over 8 h of treatment. At 1 mM metformin, lactate increasedfrom 0.30±0.1 to 3.34±0.2 over 8 h and at 10 mM metformin, lactateincreased from 0.22±0.1 to 5.76±0.7 mM. The corresponding pH droppedfrom 7.4±0.01 in both groups to 7.16±0.03 and 7.00±0.04 for 1 mM and 10mM metformin, respectively. Phenformin-treated platelets (0.5 mM)produced similar levels of lactate as 10 mM metformin-treated samples.The level of lactate increase correlated with the decrease in pH for alltreatment groups. The increased lactate levels in metformin-treatedintact platelets also correlated with decreased absolute OXPHOS_(CI)respiratory values seen in metformin-treated permeabilized platelets(r²=0.60, P<0.001). A limited set of experiments further demonstratedthat intact PBMCs also show increased lactate release upon exposure to10 mM metformin (data not shown).

Discussion of the Results from Examples 18-19

This study demonstrates a non-reversible toxic effect of metformin onmitochondria specific for complex I in human platelets and PBMCs atconcentrations relevant for the clinical condition of metforminintoxication. In platelets, we further have shown a correlation betweendecreased Complex I respiration and increased production of lactate. Themitochondrial toxicity we observed for metformin developed over time inintact cells. Phenformin, a structurally related compound now withdrawnin most countries due to a high incidence of LA, induced lactate releaseand pH decline in platelets through a complex I specific effect atsubstantially lower concentration.

In the present study, using a model applying high-resolutionrespirometry to assess integrated mitochondrial function of humanplatelets, we have demonstrated that the mitochondrial toxicity of bothmetformin and phenformin is specific to respiratory complex I and that asimilar specific inhibition also is present in PBMCs. Complex Irespiration of permeabilized PBMCs was 2.6-fold more sensitive tometformin than that of permeabilized platelets. However, due to thetime-dependent toxicity of metformin (see below), the IC₅₀ is possiblyan underestimation and could be lower if determined after longerexposure time. These findings further strengthen that the mitochondrialtoxicity of metformin is not limited to specific tissues, as shownpreviously by others, but rather a generalized effect on a subcellularlevel. The metformin-induced complex IV inhibition in platelets reportedby (Protti et al., 2012a, Protti et al., 2012b) has not been confirmedin this study or in an earlier study by Dykens et al. (2008) usingisolated bovine mitochondria. Further, metformin and phenformin did notinduce respiratory inhibition through any unspecific permeabilitychanges of the inner or outer mitochondrial membranes as there were noevidence of uncoupling or stimulatory response following cytochrome caddition in presence of the drugs. High-resolution respirometry is amethod of high sensitivity and allows O₂ measurements in the picomolarrange. When applied to human blood cells ex vivo, it allows assessmentof respiration in the fully-integrated state in intact cells, andpermits exogenous supply and control of substrates to intactmitochondria in permeabilized cells. This is in contrast to enzymaticspectrophotometric assays which predominantly have been used in theresearch on mitochondrial toxicity of metformin, for instance by D kenset al. (2008) and Owen et al. 2000). These assays measure theindependent, not-integrated function of the single complexes and hence,are less physiological, which may contribute to the differences inresults between our studies.

The results of the study demonstrated significant respiratoryinhibition, lactate increase and pH decrease in intact plateletsuspensions caused by metformin at concentrations relevant forintoxication already after 8-18 h. The time-dependent inhibition ofmitochondrial respiration in combination with the lack of reversalfollowing exchange of the extracellular buffer and dilution ofintracellular content of soluble metformin by permeabilization of thecell point towards intramitochondrial accumulation being a key factor inthe development of drug-induced mitochondrial dysfunction-related LA, ashas been proposed by others (Chan et al., 2005, Lalau, 2010).

Phenformin's mitochondrial toxicity has been shown previously, forinstance on HepG2 cells, a liver carcinoma cell line, and isolatedmitochondria of rat and cow. Here we have demonstrated specificmitochondrial toxicity also using human blood cells. Compared tometformin, phenformin had a stronger mitochondrial toxic potency onhuman platelets (IC₅₀ 1.2 mM and 0.058 mM, respectively). Phenformin andmetformin show a 10 to 15-fold difference in clinical dosing and 3 to10-fold difference in therapeutic plasma concentration. In this study wehave observed a 20-fold difference between phenformin and metformin inthe potential to inhibit complex I. If translated to patients thisdifference in mitochondrial toxicity in relation to clinical dosingcould potentially explain phenformin's documented higher incidence ofphenformin-associated LA.

Standard therapeutic plasma concentrations of metformin are in the rangeof 0.6 and 6.0 μM and toxic concentrations lie between 60 μM and 1 mM.In a case report of involuntary metformin intoxication, prior tohemodialysis, a serum level of metformin over 2 mM was reported (Al-Abriet al., 2013). Tissue distribution studies have further demonstratedthat the metformin concentration under steady-state is lower inplasma/serum than in other organs. It has been shown to accumulate in 7to 10-fold higher concentrations in the gastrointestinal tract, withlesser but still significantly higher amounts in the kidney, liver,salivary glands, lung, spleen and muscle as compared to plasma levels.Under circumstances where the clearance of metformin is impaired, suchas predisposing conditions affecting the cardiovascular system, liver orkidneys, toxic levels can eventually be reached. The toxic concentrationof metformin seen in the present study (1 mM) is thus comparable to whatis found in the blood of metformin-intoxicated patients. Althoughmetformin is toxic to blood cells, as shown in this study, it isunlikely that platelets and PBMCs are major contributors to thedevelopment of LA. As metformin is accumulated in other organs andadditionally these organs are more metabolically active, increasedlactate production is likely to be seen first in other tissues. Ourresults therefore strengthen what has been suggested by others (Brunmairet al., 2004, Protti et al., 2012b, Dykens et al., 2008), that systemicmitochondrial inhibition is the cause of metformin-induced LA.

Based on earlier studies and the present findings it is intriguing tospeculate on the possibility that metformin's anti-diabetic effect maybe related to inhibition of aerobic respiration. The decreased glucoselevels in the liver and decreased uptake of glucose to the blood in thesmall intestine in metformin-treated diabetic patients might be due topartial complex I inhibition. Complex I inhibition causes reducedproduction of ATP, increased amounts of AMP, activation of the enzymeAMP-activated protein kinase (AMPK), and accelerated glucose turnover byincreased glycolysis, trying to compensate for the reduced ATPproduction.

Until now, treatment measures for metformin-associated LA consist ofhaemodialysis and haemofiltration to remove the toxin, correct for theacidosis and increase renal blood flow.

Example 20

Intervention on Metformin-Induced Increase in Lactate Production withCell-Permeable Succinate Prodrugs

Intervention of metformin-induced increase in lactate production inintact human platelets with newly developed and synthesizedcell-permeable succinate prodrugs was done in PBS containing 10 mMglucose. The platelets were exposed to either rotenone alone (2 μM),rotenone (2 μM) and antimycin (1 μg/mL, only for cells treated with NV189), or 10 mM metformin and after 60 min either vehicle (DMSO,control), either of the cell-permeable succinate prodrugs (NV118, NV189and NV241), or succinate were added at a concentration of 250 μM each 30minutes. Lactate levels were measured in intervals of 30 min with theonset of the experiment. Additionally, pH was measured prior to thefirst addition of vehicle (dmso, control), the different cell-permeablesuccinate prodrugs (NV118, NV189, NV241) or succinate and at the end ofthe experiment. The rate of lactate production was calculated with anonlinear fit with a 95% Confidence interval (CI) of the lactate-timecurve slope (FIGS. 9, 10, 11 and 12).

Results relating to Example 20 are based on the assays described herein.

Lactate Production Due to Rotenone and Metformin Incubation inThrombocytes is Attenuated by the Addition of Cell-Permeable SuccinateProdrugs

The rate of lactate production in thrombocytes incubated with 2 μMRotenone was 0.86 mmol lactate (200·10⁶trc·h)⁻¹ (95% ConfidenceInterval) [CI] 0.76-0.96) which was attenuated by NV118 (0.25 mmol [95%CI 0.18-0.33]), NV189 (0.42 mmol [95% CI 0.34-0.51]) and NV241 (0.34mmol [95% CI 0.17-0.52]), which was not significantly different fromcells not receiving rotenone (0.35 [95% CI 0.14-0.55]) (FIGS. 9,10 and11). Cells incubated with antimycin in addition to rotenone and NV189had a lactate production comparable to rotenone-treated cell (0.89 mmol[0.81-0.97]), demonstrating the specific mitochondrial effect of thecell-permeable succinate prodrugs (FIG. 10). Cells incubated with 10 mMMetformin produce lactate at a rate of 0.86 mmol lactate (200·10⁹trc·h)⁻¹ (95% CI 0.69-1.04) compared 0.22 mmol (95% CI 0.14-0.30) invehicle (water) treated cells (FIG. 12). Co-incubating with either ofthe three succinate prodrugs attenuate the metformin effect resulting in0.43 mmol production (95% CI 0.33-0.54) for NV118 (FIG. 9), 0.55 mmol(95% CI 0.44-0.65) for NV189 (FIG. 10), and 0.43 mmol (95% CI 0.31-0-54)for NV241 (FIG. 11).

All references referred to in this application, including patent andpatent applications, are incorporated herein by reference to the fullestextent possible.

Throughout the specification and the claims which follow, unless thecontext requires otherwise, the word ‘comprise’, and variations such as‘comprises’ and ‘comprising’, will be understood to imply the inclusionof a stated integer, step, group of integers or group of steps but notto the exclusion of any other integer, step, group of integers or groupof steps.

The application of which this description and claims forms part may beused as a basis for priority in respect of any subsequent application.The claims of such subsequent application may be directed to any featureor combination of features described herein. They may take the form ofproduct, composition, process, or use claims and may include, by way ofexample and without limitation, the following claims:

All references referred to in this application, including patent andpatent applications, are incorporated herein by reference to the fullestextent possible.

Throughout the specification and the claims which follow, unless thecontext requires otherwise, the word ‘comprise’, and variations such as‘comprises’ and ‘comprising’, will be understood to imply the inclusionof a stated integer, step, group of integers or group of steps but notto the exclusion of any other integer, step, group of integers or groupof steps. The word “comprise” includes “contain” and “consist of”.

General Description of the Class of Compounds to which the CompoundsAccording to the Invention Belong and Specific Embodiments

The class of compounds may be defined by formula (IB) below,

or a pharmaceutically acceptable salt thereof. Where the dotted bondbetween A and B denotes an optional bond so as to form a ring closedstructure, wherein

Z is selected from —CH₂—CH₂— or >CH(CH₃), —O, S,

A and B are independently different or identical and are selected from—O—R′, —NHR″, —SR′″ or —OH, with the proviso that both A and B cannot beH,

R′, R″ and R′″ are independently different or identical and selectedfrom the formula (IIB) to (IXB) below:

R₁=H, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, O-acyl,O-alkyl, N-acyl, N-alkyl, Xacyl, CH₂Xalkyl, CH₂X-acyl, F, CH₂COOH,CH₂CO₂alkyl or any of the below formulas (a)-(f)

In preferred structures, R₁=H, Me, Et, propyl, i-propyl, butyl,iso-butyl, t-butyl, O-acyl, O-alkyl, N-acyl, N-alkyl, Xacyl, CH₂Xalkyl,CH₂X-acyl, F, CH₂COOH.

X═O, NH, NR₆, S

R₂=Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, —C(O)CH₃,—C(O)CH₂C(O)CH₃, —C(O)CH₂CH(OH)CH₃,

R₃=R₁, i.e. is the same or different groups as mentioned under R₁═CR′₃R′₃, NR₄

n=1-4,

p=1-2

X₂=OR5, NR₁R′₂

R′₃=H, Me, Et, F

R₄=H, Me, Et, i-Pr

R₅=acetyl, propionyl, benzoyl, benzylcarbonyl

R′₂=H.HX₃, acyl, acetyl, propionyl, benzoyl, benzylcarbonyl

X₃=F, Cl, Br and I

R₆=H, or alkyl such as e.g. Me, Et, n-propyl, i-propyl, butyl,iso-butyl, t-butyl, or acetyl, such as e.g. acyl, propionyl, benzoyl, orformula (IIB), formula (IIBI) or formula (VIIIB)

X₅=—H, —COOH, —C(═O)XR₆,

X₅ may also be CONR₁R₃.

R₉=H, Me, Et or O₂CCH₂CH₂COXR₈

R₁₀=Oacyl, NHalkyl, NHacyl, or O₂CCH₂CH₂COX₆R₈

X₆=O, NR₈

R₈=H, alkyl, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl,acetyl, acyl, propionyl, benzoylor formula (IIB),

R₁₁ and R₁₂ are independently H, alkyl, Me, Et, propyl, i-propyl, butyl,iso-butyl, t-butyl, acetyl, acyl, propionyl, benzoyl, acyl, —CH₂Xalkyl,—CH₂Xacyl, where X=O, NR₆ or S,

R_(c) and R_(d) are independently CH₂Xalkyl, CH₂Xacyl, where X=O, NR₆ orS,

Rf, Rg and Rh are independently selected from Xacyl, —CH₂Xalkyl,—CH₂X—acyl and R₉,

wherein alkyl is e.g. methyl, ethyl, propyl, isopropyl, n-butyl,isobutyl, sec-butyl, tert-butyl, n-pentyl, neopentyl, isopentyl, hexyl,isohexyl, heptyl, octyl, nonyl or decyl and acyl is e.g. formyl, acetyl,propionyl, butyryl pentanoyl, benzoyl and the like, and wherein theacyls and alkyls may be optionally substituted,

the dotted bond between A and B denotes an optional bond to form acyclic structure of formula (I) and with the proviso that when such acyclic bond is present, the compound according to formula (I) isselected from

wherein X₄ is selected from —COOH, —C(═O)XR₆,

and wherein R_(x) and R_(y) are independently selected from R₁, R₂, R₆or R′, R″ or R′″ with the proviso that R_(x) and R_(y) cannot both be—H.

In preferred aspect, R′, R″ and R′″ are independently different oridentical and selected from the formula (IIB), (VB), (VIIB) or VIIIB)below:

Preferably, and with respect to formula (IIB), at least one of R₁ and R₃is —H, such that formula II is:

Preferably, and with respect to formula (VII), p is 1 or 2, preferably pis 1 and X₅ is —H such that formula (VIIB) is

Preferably, and with respect to formula (IXB), at least one of R_(f),R_(g), R_(h) is —H or alkyl, with alkyl as defined herein. Moreover, itis also preferable with respect to Formula (IXB) that at least one ofRf, Rg, Rh is —CH₂Xacyl, with acyl as defined herein.

An interesting subclass of the class mentioned above relates to thecompounds of Formula (I)

or a pharmaceutically acceptable salt thereof. The dotted bond between Aand B denotes an optional bond so as to form a ring closed structure.

In formula (IC) Z is selected from —CH₂—CH₂— or >CH(CH₃),

A is selected from —SR, —OR and NHR, and wherein R is

B is selected from —O—R′, —NHR″, —SR′″ or —OH; R′ is selected from theformula (IIC) to (IXC) below:

Preferably, R′ is selected from the formula (IIC), (VC), to (IXC) below:

R′, R″ and R′″ are independently different or identical and is selectedfrom formula (IVC-VIIIC) below:

R₁=H, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, O-acyl,O-alkyl, N-acyl, N-alkyl, Xacyl, CH₂Xalkyl, CH₂X-acyl, F, CH₂COOH,CH₂CO₂alkyl or any of formulae (a)-(f)

Preferably, R₁=H, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl,O-acyl, O-alkyl, Nacyl, N-alkyl, Xacyl, CH₂Xalkyl, CH₂X-acyl, F,CH₂COOH, CH₂CO₂alkyl,

X=O, NH, NR₆, S

R₂=Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, C(O)CH₃,C(O)CH₂C(O)CH₃, C(O)CH₂CH(OH)CH₃,

R₃=R₁, i.e. may be the same or a different group as defined under R₁,

X₁=CR′₃R′₃, NR₄

n=1-4,

p=1-2

X₂=OR₅, NR₁R′₂

R′₃=H, Me, Et, F

R₄=H, Me, Et, i-Pr

R₅=acetyl, propionyl, benzoyl, benzylcarbonyl

R′₂=H.HX₃, acyl, acetyl, propionyl, benzoyl, benzylcarbonyl

X₃=F, Cl, Br and I

R₆=H, alkyl, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl,acetyl, acyl, propionyl, benzoyl, or formula (IIC), formula (IIIC) orformula (VIIIC)

X₅=—H, —COOH, —C(═O)XR₆,

X₅ may also be CONR₁R₃

R₉=H, Me, Et or O₂CCH₂CH₂COXR₈

R₁₀=Oacyl, NHalkyl, NHacyl, or O₂CCH₂CH₂COX₆R₈

X₆=O, NR₈

R₈=H, alkyl, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl,acetyl, acyl, propionyl, benzoyl, or formula (IIC), formula (IIIC) orformula (VIIIC)

R₁₁ and R₁₂ are independently H, alkyl, Me, Et, propyl, i-propyl, butyl,iso-butyl, t-butyl, acetyl, acyl, propionyl, benzoyl, acyl, —CH₂Xalkyl,—CH₂Xacyl, where X=O, NR₆ or S

R_(c) and R_(d) are independently CH₂Xalkyl, CH₂Xacyl, where X=O, NR₆ orS,

R_(f), R_(g) and R_(h) are independently selected from Xacyl,—CH₂Xalkyl, —CH₂X—acyl and R₉

alkyl is e.g. Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl andacyl is e.g. formyl, acetyl, propionyl, isopropionyl, byturyl,tert-butyryl, pentanoyl, benzoyl and the likes and wherein the acyls andalkyls may be optionally substituted, and

when the dotted bond between A and B is present, the compound accordingto formula (I) is

wherein X₄ is selected from —COOH, —C(═O)XR₆,

Preferably, and with respect to formula (IIC), at least one of R₁ and R₃is —H, such that formula II is:

Preferably, and with respect to formula (VIIC), p is 1 or 2, preferablyp is 1 and X₅ is —H such that formula (VIIC) is

Preferably, and with respect to formula (IXC), at least one of R_(f),R_(g), R_(h) is —H or alkyl, with alkyl as defined herein. Moreover, itis also preferable with respect to Formula (IXC) that at least one ofR_(f), R_(g), R_(h) is —CH₂Xacyl, with acyl as defined herein.

Interesting compounds according to formula (IC) are:

-   -   wherein X₄ is selected from —COOH, —C(═O)XR₆,

-   -   wherein R₁ and X₅ is as defined herein. Preferably X₅ is —H.

-   -   wherein R₆, X₅ and R₁ are as defined herein. Preferably X₅ is        —H.

-   -   wherein X₅ and R₁ are as defined herein.

Preferably X₅ is —H.

Specific Embodiments

1. A compound according to Formula (I)

or a pharmaceutically acceptable salt thereof, wherein the dotted bondbetween A and B denotes an optional bond so as to form a ring closedstructure, and wherein

Z is selected from —CH₂—CH₂— or >CH(CH₃),

A is selected from —SR, —OR and NHR and R is

B is selected from —O—R′, —NHR″, —SR′″ or —OH; and R′ is selected fromthe formula (II) to (IX) below:

R′, R″ and R′″ are independently different or identical and is selectedfrom formula (IV-VIII) below:

R₁=H, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, O-acyl,O-alkyl, N-acyl, N-alkyl, Xacyl, CH₂Xalkyl, CH₂X-acyl, F, CH₂COOH,CH₂CO₂alkyl or any of the below formulae (a)-(f)

X=O, NH, NR₆, S

R₂=Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, C(O)CH₃,C(O)CH₂C(O)CH₃, C(O)CH₂CH(OH)CH₃,

R₃=R₁, i.e. different or identical with the groups mentioned under R₁,

X₁=CR′₃R′₃, NR₄

n=1-4,

p=1-2

X₂=OR₅, NR₁R′₂

R′₃=H, Me, Et, F

R₄=H, Me, Et, i-Pr

R₅=acetyl, propionyl, benzoyl, benzylcarbonyl

R′₂=H.HX₃, acyl, acetyl, propionyl, benzoyl, benzylcarbonyl

X₃=F, Cl, Br and I

R₆=H, alkyl, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl,acetyl, acyl, propionyl, benzoyl, or formula (II), formula (III) orformula (VIII)

X₅=—H, —COOH, —C(═O)XR₆,

R₉=H, Me, Et or O₂CCH₂CH₂COXR₈

R₁₀=Oacyl, NHalkyl, NHacyl, or O₂CCH₂CH₂CO X₆R₈

X₆=O, NR₈

R₈=H, alkyl, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl,acetyl, acyl, propionyl, benzoyl, or formula (II), formula (III) orformula (VIII)

R₁₁ and R₁₂ are independently H, alkyl, Me, Et, propyl, i-propyl, butyl,iso-butyl, t-butyl, acetyl, acyl, propionyl, benzoyl, acyl, —CH₂Xalkyl,—CH₂Xacyl, where X=O, NR₆ or S

R_(c) and R_(d) are independently CH₂Xalkyl, CH₂Xacyl, where X=O, NR₆ orS,

R_(f), R_(g) and R_(h) are independently selected from Xacyl,—CH₂Xalkyl, —CH₂X—acyl and R₉

alkyl is e.g. Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl and

acyl is e.g. formyl, acetyl, propionyl, isopropionyl, byturyl,tert-butyryl, pentanoyl, benzoyl and the likes and wherein the acyls oralkyls may be optionally substituted, and when the dotted bond between Aand B is present, the compound according to formula (I) is

wherein X₄ is selected from —COOH, —C(═O)XR₆,

and with the further proviso that the compound is not any of the belowcompounds

2. A compound according to embodiment 1, wherein formula (II) is suchthat at least one of R1 and R₃ is —H such that formula II is:

3. A compound according to embodiment 1, wherein formula (III) is suchthat R₄ is —H and formula (III) is

and X₁ is NH

4. A compound according to embodiment 1, wherein formula (VII) is suchthat, p=2 and X₅ is —H and formula (VII) is

5. A compound according to embodiment 1, wherein formula (IX) is suchthat at least one of R_(f), R_(g), R_(h) is —H or alkyl, with alkyl asdefined herein.

6. A compound according to embodiment 1 or 5, wherein formula (IX) issuch that at least one of Rf, Rg, Rh is —CH₂Xacyl, with acyl as definedherein.

7. A compound according to any of embodiments 1-6, wherein Formula (I)is

wherein X₄ is selected from —COOH, —C(═O)XR₆,

8. A compound according to any of embodiments 1-6, wherein Formula (I)is

Wherein X₅ and R₁ is as defined in claim 1 and wherein X₅ is preferably—H

9. A compound according to any of embodiments 1-6, wherein Formula (I)is

wherein X₅ and R₁ is as defined in embodiment 1 and wherein X₅ ispreferably —H

10. A compound according to any of embodiments 1-6, wherein Formula (I)is

Wherein X₅, R₁ and R₆ is as defined in embodiment 1 and wherein X₅ ispreferably —H.

11. A compound according to any of embodiments 1-10 for use in medicine

12. A compound according to any of embodiments 1-10, for use incosmetics

13. A compound according to any of embodiments 1-10 for use in thetreatment of or prevention of metabolic diseases, or in the treatment ofdiseases of mitochondrial dysfunction or disease related tomitochondrial dysfunction, treating or suppressing of mitochondrialdisorders, stimulation of mitochondrial energy production, treatment ofcancer and following hypoxia, ischemia, stroke, myocardial infarction,acute angina, an acute kidney injury, coronary occlusion and atrialfibrillation, or to avoid or counteract reperfusion injuries.

14. A compound according for use according to embodiment 11, wherein themedical use is prevention or treatment of drug-induced mitochondrialside-effects.

15. A compound for use according to embodiment 14, wherein theprevention or drug-induced mitochondrial side-effects relates to druginteraction with Complex I, such as e.g. metformin-Complex Iinteraction.

16. A compound according to embodiment 13, wherein diseases ofmitochondrial dysfunction involves e.g. mitochondrial deficiency such asa Complex I, II, III or IV deficiency or an enzyme deficiency like e.g.pyruvate dehydrogenase deficiency.

17. A compound for use according to any of embodiments 13-16, whereinthe diseases of mitochondrial dysfunction or disease related tomitochondrial dysfunction are selected from Alpers Disease (ProgressiveInfantile Poliodystrophy, Amyotrophic lateral sclerosis (ALS), Autism,Barth syndrome (Lethal Infantile Cardiomyopathy), Beta-oxidationDefects, Bioenergetic metabolism deficiency, Carnitine-Acyl-CarnitineDeficiency, Carnitine Deficiency, Creatine Deficiency Syndromes(Cerebral Creatine Deficiency Syndromes (CCDS) includes:Guanidinoaceteate Methyltransferase Deficiency (GAMT Deficiency),L-Arginine:Glycine Amidinotransferase Deficiency (AGAT Deficiency), andSLC6A8-Related Creatine Transporter Deficiency (SLC6A8 Deficiency),Co-Enzyme Q10 Deficiency, Complex I Deficiency (NADH dehydrogenase(NADHCoQ reductase deficiency), Complex II Deficiency (Succinatedehydrogenase deficiency), Complex III Deficiency (Ubiquinone-cytochromec oxidoreductase deficiency), Complex IV Deficiency/COX Deficiency(Cytochrome c oxidase deficiency is caused by a defect in Complex IV ofthe respiratory chain), Complex V Deficiency (ATP synthase deficiency),COX Deficiency, CPEO (Chronic Progressive External OphthalmoplegiaSyndrome), CPT I Deficiency, CPT II Deficiency, Friedreich's ataxia(FRDA or FA), Glutaric Aciduria Type II, KSS (Kearns-Sayre Syndrome),Lactic Acidosis, LCAD (Long-Chain Acyl-CoA Dehydrogenase Deficiency),LCHAD, Leigh Disease or Syndrome (Subacute NecrotizingEncephalomyelopathy), LHON (Leber's hereditary optic neuropathy), LuftDisease, MCAD (Medium-Chain Acyl-CoA Dehydrogenase Deficiency), MELAS(Mitochondrial Encephalomyopathy Lactic Acidosis and StrokelikeEpisodes), MERRF (Myoclonic Epilepsy and Ragged-Red Fiber Disease),MIRAS (Mitochondrial Recessive Ataxia Syndrome), MitochondrialCytopathy, Mitochondrial DNA Depletion, Mitochondrial Encephalopathyincluding: Encephalomyopathy and Encephalomyelopathy, MitochondrialMyopathy, MNGIE (Myoneurogastointestinal Disorder and Encephalopathy,NARP (Neuropathy, Ataxia, and Retinitis Pigmentosa), Neurodegenerativedisorders associated with Parkinson's, Alzheimer's or Huntington'sdisease, Pearson Syndrome, Pyruvate Carboxylase Deficiency, PyruvateDehydrogenase Deficiency, POLG Mutations, Respiratory ChainDeficiencies, SCAD (Short-Chain Acyl-CoA Dehydrogenase Deficiency),SCHAD, VLCAD (Very Long-Chain Acyl-CoA Dehydrogenase Deficiency).

18. A compound for use according to embodiment 17, wherein themitochondrial dysfunction or disease related to mitochondrialdysfunction is attributed to complex I dysfunction and selected fromLeigh Syndrome, Leber's hereditary optic neuropathy (LHON), MELAS(mitochondrial encephalomyopathy, lactic acidosis, and stroke-likeepisodes) and MERRF (myoclonic epilepsy with ragged red fibers).

19. A composition comprising a compound of Formula (I) as definedaccording any of embodiments 1-10 and one or more pharmaceutically orcosmetically acceptable excipients.

20. A method of treating a subject suffering from diseases ofmitochondrial dysfunction or disease related to mitochondrialdysfunction as defined in any of embodiments 16-18, the methodcomprising administering to the subject an efficient amount of acomposition as defined in embodiment 19.

21. A method according to embodiment 20, wherein the composition isadministered parenterally, orally, topically (including buccal,sublingual or transdermal), via a medical device (e.g. a stent), byinhalation or via injection (subcutaneous or intramuscular) 22. A methodaccording to any of embodiments 20-21, wherein the composition isadministered as a single dose or a plurality of doses over a period oftime, such as e.g. one daily, twice daily or 3-5 times daily as needed.

23. A compound according to any of embodiments 1-10 for use in thetreatment or prevention of lactic acidosis.

24. A compound according to any of embodiments 1-10 for use in thetreatment or prevention of a drug-induced side-effect selected fromlactic acidosis and side-effects related to Complex I defect, inhibitionor malfunction.

25. A compound according to any of embodiments 1-10 for use in thetreatment or prevention of a drug-induced side-effect selected fromlactic acidosis and side-effects related to defect, inhibition ormal-function in aerobic metabolism upstream of complex I (indirectinhibition of Complex I, which would encompass any drug effect thatlimits the supply of NADH to Complex I, e.g. effects on Krebs cycle,glycolysis, beta-oxidation, pyruvate metabolism and drugs that affectthe levels of glucose or other Complex I-related substrates).

26. A combination of a drug substance and a compound according to any ofembodiments 1-10 for use in the treatment and/or prevention of adrug-induced side-effect selected from i) lactic acidosis, ii) andside-effects related to a Complex I defect, inhibition or malfunction,and iii) side-effects related to defect, inhibition or malfunction inaerobic metabolism upstream of complex I (indirect inhibition of ComplexI, which would encompass any drug effect that limits the supply of NADHto Complex I, e.g. effects on Krebs cycle, glycolysis, beta-oxidation,pyruvate metabolism and drugs that affect the levels of glucose or otherComplex-I-related substrates), wherein

i) the drug substance is used for treatment of a disease for which thedrug substance is indicated, and

ii) the succinate prodrug is used for prevention or alleviation of theside effects induced or inducible by the drug substance, wherein theside-effects are selected from lactic acidosis and side-effects relatedto a Complex I defect, inhibition or malfunction.

27. A composition comprising a drug substance and a compound accordingto any of embodiments 1-10, wherein the drug substance has a potentialdrug-induced side-effect selected from i) lactic acidosis, ii)side-effects related to a Complex I defect, inhibition or malfunction,and iii) side-effects related to defect, inhibition or malfunction inaerobic metabolism upstream of complex I (indirect inhibition of ComplexI, which would encompass any drug effect that limits the supply of NADHto Complex I, e.g. effects on Krebs cycle, glycolysis, beta-oxidation,pyruvate metabolism and even drugs that affect the levels of glucose orother Complex-I-related substrates).

28. A kit comprising

i) a first container comprising a drug substance, which has a potentialdrug-induced side-effect selected i) from lactic acidosis, ii) andside-effects related to a Complex I defect, inhibition or malfunction,and iii) side-effects related to defect, inhibition or malfunction inaerobic metabolism upstream of complex I (indirect inhibition of ComplexI, which would encompass any drug effect that limits the supply of NADHto Complex I, e.g. effects on Krebs cycle, glycolysis, beta-oxidation,pyruvate metabolism and even drugs that affect the levels of glucose orother substrates), and

ii) a second container comprising a compound according to any ofembodiments 1-10, which has the potential for prevention or alleviationof the side effects induced or inducible by the drug substance, whereinthe side-effects are selected from i) lactic acidosis, ii) side-effectsrelated to a Complex I defect, inhibition or malfunction, and iii)side-effects related to defect, inhibition or malfunction in aerobicmetabolism upstream of complex I (indirect inhibition of Complex I,which would encompass any drug effect that limits the supply of NADH toComplex I, e.g. effects on Krebs cycle, glycolysis, beta-oxidation,pyruvate metabolism and even drugs that affect the levels of glucose orother substrates).

29. A method for treating a subject suffering from a drug-inducedside-effect selected from i) lactic acidosis, ii) side-effect related toa Complex I defect, inhibition or malfunction, and iii) side-effectsrelated to defect, inhibition or malfunction in aerobic metabolismupstream of complex I (indirect inhibition of Complex I, which wouldencompass any drug effect that limits the supply of NADH to Complex I,e.g. effects on Krebs cycle, glycolysis, beta-oxidation, pyruvatemetabolism and even drugs that affect the levels of glucose or othersubstrates, the method comprises administering an effective amount of acompound according to any of embodiments 1-10 to the subject.

30. A method for preventing or alleviating a drug-induced side-effectselected from i) lactic acidosis, ii) side-effect related to a Complex Idefect, inhibition or malfunction, and iii) side-effects related todefect, inhibition or malfunction in aerobic metabolism upstream ofcomplex I (indirect inhibition of Complex I, which would encompass anydrug effect that limits the supply of NADH to Complex I, e.g. effects onKrebs cycle, glycolysis, beta-oxidation, pyruvate metabolism and evendrugs that affect the levels of glucose or other substrates) in asubject, who is suffering from a disease that is treated with a drugsubstance, which potentially induce a side-effect selected from i)lactic acidosis, ii) side-effect related to a Complex I defect,inhibition or malfunction, and iii) side-effects related to defect,inhibition or malfunction in aerobic metabolism upstream of Complex I,such as in dehydrogenases of Kreb's cycle, pyruvate dehydrogenase andfatty acid metabolism, the method comprises administering an effectiveamount of a compound according to any of embodiments 1-10 to thesubject.

31. A method according to any one of embodiments 29-30, wherein the drugsubstance is an anti-diabetic substance.

32. A method according to any one of embodiments 29-31, wherein theanti-diabetic substance is metformin.

33. A compound according to any of embodiments 1-10, for use in thetreatment of absolute or relative cellular energy deficiency.

1. A method for treating or preventing metabolic diseases, treatingdiseases of mitochondrial dysfunction or disease related tomitochondrial dysfunction, treating or suppressing of mitochondrialdisorders, stimulating mitochondrial energy production, treating cancer,hypoxia, ischemia, stroke, myocardial infarction, acute angina, an acutekidney injury, coronary occlusion or atrial fibrillation, or avoiding orcounteracting reperfusion injuries, said method comprising administeringto a subject a compound of Formula (I)

or a pharmaceutically acceptable salt thereof, wherein the dotted bondbetween A and B denotes an optional bond so as to form a ring closedstructure, and wherein Z is selected from —CH₂—CH₂— or >CH(CH₃), A isselected from —SR, —OR and NHR, and R is

B is selected from —O—R′, —NHR″, —SR′″ or —OH; and R′ is selected fromthe formulas below:

R′, R″ and R′″ are independently different or identical and is selectedfrom the formulas below:

R₁ and R3 are independently different or identical and are selected fromH, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, O-acyl, O-alkyl,N-acyl, N-alkyl, Xacyl, CH₂Xalkyl, CH₂X-acyl, F, CH₂COOH, CH₂CO₂alkyl, Xis selected from O, NH, NR₆, S, R₂ is selected from Me, Et, propyl,i-propyl, butyl, iso-butyl, t-butyl, C(O)CH₃, C(O)CH₂C(O)CH₃,C(O)CH₂CH(OH)CH₃, p is an integer and is 1 or 2 R₆ is selected from H,Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, acetyl, acyl,propionyl, benzoyl, or formula (II), or formula (VIII) X₅ is selectedfrom —H, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, —COOH,—C(═O)XR₆, CONR₁R₃ or is formula

X₇ is selected from R₁, —NR₁R₃, R₉ is selected from H, Me, Et orO₂CCH₂CH₂COXR₈ R₁₀ is selected from Oacyl, NHalkyl, NHacyl, orO₂CCH₂CH₂COX₆R₈ X₆ is selected from O, NR₈, NR₆R₈, wherein R₆ and R₈ areindependently different or identical and are is selected from H, alkyl,Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, acetyl, acyl,propionyl, benzoyl, or formula (II), or formula (VIII), R₁₁ and R₁₂ areindependently different or identical and are selected from H, Me, Et,propyl, i-propyl, butyl, iso-butyl, t-butyl, acetyl, propionyl, benzoyl,—CH₂Xalkyl, —CH₂Xacyl, where X is O, NR₆ or S, R_(c) and R_(d) areindependently different or identical and are selected from CH₂Xalkyl,CH₂Xacyl, where X=O, NR₆ or S, R₁₃, R₁₄ and R₁₅ are independentlydifferent or identical and are selected from H, Me, Et, propyl,i-propyl, butyl, iso-butyl, t-butyl, —COOH, O-acyl, O-alkyl, N-acyl,N-alkyl, Xacyl, CH₂Xalkyl; substituents on R13 and R14 or R13 and R15may bridge to form a cyclic system, R_(f), R_(g) and R_(h) areindependently different or identical and are selected from Xacyl,—CH₂Xalkyl, —CH₂X—acyl and R₉, alkyl is selected from Me, Et, propyl,i-propyl, butyl, iso-butyl, t-butyl, acyl is selected from formyl,acetyl, propionyl, isopropionyl, butyryl, tert-butyryl, pentanoyl,benzoyl, acyl and/or alkyl may be optionally substituted, and when thedotted bond between A and B is present, the compound according toformula (I) is

wherein X₄ is selected from —COOH, —C(═O)XR₆,


2. The method according to claim 1, for preventing or treatingdrug-induced mitochondrial side-effects.
 3. The method according toclaim 2, wherein the drug-induced mitochondrial side-effects relates todrug interaction with Complex I.
 4. The method according to claim 1,wherein diseases of mitochondrial dysfunction or disease related tomitochondrial dysfunction involve Complex I, II, III or IV deficiency oran enzyme deficiency.
 5. The method according to claim 1, wherein thediseases of mitochondrial dysfunction or disease related tomitochondrial dysfunction are selected from the group consisting ofAlpers Disease (Progressive Infantile Poliodystrophy), Amyotrophiclateral sclerosis (ALS), Autism, Barth syndrome (Lethal InfantileCardiomyopathy), Beta-oxidation Defects, Bioenergetic metabolismdeficiency, Carnitine-Acyl-Carnitine Deficiency, Carnitine Deficiency,Creatine Deficiency Syndromes, Cerebral Creatine Deficiency Syndromes(CCDS), Guanidinoaceteate Methyltransferase Deficiency (GAMTDeficiency), L-Arginine:Glycine Amidinotransferase Deficiency (AGATDeficiency), and SLC6A8-Related Creatine Transporter Deficiency (SLC6A8Deficiency), Co-Enzyme Q10 Deficiency Complex I Deficiency (NADHdehydrogenase (NADH-CoQ reductase deficiency), Complex II Deficiency(Succinate dehydrogenase deficiency), Complex III Deficiency(Ubiquinone-cytochrome c oxidoreductase deficiency), Complex IVDeficiency/COX Deficiency (Cytochrome c oxidase deficiency, Complex VDeficiency (ATP synthase deficiency), COX Deficiency, CPEO (ChronicProgressive External Ophthalmoplegia Syndrome), CPT I Deficiency, CPT IIDeficiency, Friedreich's ataxia (FRDA or FA), Glutaric Aciduria Type II,KSS (Kearns-Sayre Syndrome), Lactic Acidosis, LCAD (Long-Chain Acyl-CoADehydrogenase Deficiency), LCHAD, Leigh Disease or Syndrome (SubacuteNecrotizing Encephalomyelopathy), LHON (Leber's hereditary opticneuropathy), Luft Disease, MCAD (Medium-Chain Acyl-CoA DehydrogenaseDeficiency), MELAS (Mitochondrial Encephalomyopathy Lactic Acidosis andStrokelike Episodes), MERRF (Myoclonic Epilepsy and Ragged-Red FiberDisease), MIRAS (Mitochondrial Recessive Ataxia Syndrome), MitochondrialCytopathy, Mitochondrial DNA Depletion, Mitochondrial Encephalopathyincluding: Encephalomyopathy and Encephalomyelopathy, MitochondrialMyopathy, MNGIE (Myoneurogastointestinal Disorder and Encephalopathy,NARP (Neuropathy, Ataxia, and Retinitis Pigmentosa), Neurodegenerativedisorders associated with Parkinson's, Alzheimer's or Huntington'sdisease, Pearson Syndrome, Pyruvate Carboxylase Deficiency, PyruvateDehydrogenase Deficiency, POLG Mutations, Respiratory ChainDeficiencies, SCAD (Short-Chain Acyl-CoA Dehydrogenase Deficiency),SCHAD, and VLCAD (Very Long-Chain Acyl-CoA Dehydrogenase Deficiency). 6.The method according to claim 5, wherein the mitochondrial dysfunctionor disease related to mitochondrial dysfunction is a complex Idysfunction selected from the group consisting of Leigh Syndrome,Leber's hereditary optic neuropathy (LHON), MELAS (mitochondrialencephalomyopathy, lactic acidosis, and stroke-like episodes) and MERRF(myoclonic epilepsy with ragged red fibers).
 7. The method according toclaim 1 having Formula (IA)

or a pharmaceutically acceptable salt thereof, wherein Z is —CH₂—CH₂—, Ais selected from —SR, —OR and NHR, and R is

B is selected from —O—R′, —NHR″, —SR′″ or —OH; and R′, R″ and R′″ areindependently different or identical and is selected from one or theformulas below:

R₁ and R₃ are independently different or identical and are selected fromH, Me, Et, propyl, O-Me, O-Et, O-propyl, X is selected from O, NH, S, pis an integer and is 1, R₆ is selected from H, Me, Et, X₆ is selectedfrom —H, Me, Et, —COOH, —C(═O)XR₆, CONR₁R₃ X₇ is selected from R₁,—NR₁R₃, R₁₃, R₁₄ and R₁₅ are independently different or identical andare selected from H, Me, Et, propyl, i-propyl, butyl, iso-butyl,t-butyl, —COOH, O-acyl, O-alkyl, N-acyl, N-alkyl, Xacyl, CH₂Xalkyl,wherein alkyl and acyl are as defined herein before.
 8. The methodaccording to claim 1, wherein R₁₃, R₁₄ and R₁₅ are independentlydifferent or identical and are selected from H, Me, Et, —COOH.
 9. Themethod according to claim 1, wherein Z is —CH₂CH₂— and A is —SR.
 10. Themethod according to claim 1, wherein Z is —CH₂CH₂—, A is —SR, and B is—OR′, OH or S′″.
 11. The method according to claim 1, wherein Z is—CH₂CH₂—, A is —SR, B is —OR′, OH or SR′″, where R′″ is


12. The method according to claim 1, wherein B is —OR′ and R′ is


13. The method according to claim 1, wherein Z is —CH₂CH₂—, A is —SR andR is

p is 1, and B is —OR′ and R′ is


14. The method according to claim 1, wherein X₅ is H and R₁₃, R₁₄ andR₁₅ are H.
 15. The method according to claim 1, wherein Z is —CH₂CH₂—and A is SR and B is OH.
 16. The method according to claim 1, wherein Zis —CH₂CH₂—, A is NHR, B is OH and R is

and X is S.
 17. The method according to claim 1, wherein R and/or R′″ is

and p=1 and X₅ is —H.
 18. The method according to claim 1, wherein Rand/or R′″ is

and p=1 and X₅ is COXR₆.
 19. The method according to claim 1, wherein Rand/or R′″ is

and p=1 and X₅ is CONR₁R₃.
 20. The method according to claim 1, whereinthe compound is selected from the group consisting of: